Double-stranded oligonucleotides

ABSTRACT

Antisense sequences, including duplex RNAi compositions, which possess improved properties over those taught in the prior art are disclosed. The invention provides optimized antisense oligomer compositions and method for making and using the both in in vitro systems and therapeutically. The invention also provides methods of making and using the improved antisense oligomer compositions.

RELATED APPLICATIONS

[0001] This application claims the priority of U.S. provisional patentapplication No. 60/353,203, filed on Feb. 1, 2002, application No.60/436,238, filed Dec. 23, 2002, and application No. 60/438,608, filedJan. 7, 2003. This application also claims the priority of 60/353,381,filed Feb. 1, 2002. The entire contents of the aforementionedapplications are hereby expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Complementary oligonucleotide sequences are promising therapeuticagents and useful research tools in elucidating gene function. However,oligonucleotide molecules of the prior art are often subject to nucleasedegradation when applied to biological systems. Therefore, it is oftendifficult to achieve efficient inhibition of gene expression (includingprotein synthesis) using such compositions.

[0003] In order to maximize the usefulness, such as the potentialtherapeutic activity and in vitro utility, of oligonucleotides that arecomplementary to other sequences of interest, it would be of greatbenefit to improve upon the prior art oligonucleotides by designingimproved oligonucleotides having increased stability both against serumnucleases and cellular nucleases and nucleases found in other bodilyfluids.

SUMMARY OF THE INVENTION

[0004] The instant invention is based, at least in part, on thediscovery that double-stranded oligonucleotides comprising an antisenseoligonucleotide and a protector oligonucleotide, are capable ofinhibiting gene function. Thus, the invention improves the prior artantisense sequences, inter alia, by providing oligonucleotides which areresistant to degradation by cellular nucleases.

[0005] Accordingly, the invention provides optimized oligonucleotidecompositions and methods for making and using both in in vitro, and invivo systems, e.g., therapeutically.

[0006] In one aspect, the invention pertains to a double-strandedoligonucleotide composition having the structure:

[0007] where (1) N is a nucleomonomer in complementary oligonucleotidestrands of equal length and where the sequence of Ns corresponds to atarget gene sequence and (2) X and Y are each independently selectedfrom a group consisting of nothing; from about 1 to about 20 nucleotidesof 5′ overhang; from about 1 to about 20 nucleotides of 3′ overhang; anda loop structure consisting from about 4 to about 20 nucleomonomers,where the nucleomonomers are selected from the group consisting of G andA.

[0008] An “overhang” is a relatively short single-stranded nucleotidesequence on the 5′- or 3′-hydroxyl end of a double-strandedoligonucleotide molecule (also referred to as an “extension,”“protruding end,” or “sticky end”).

[0009] In one embodiment, the number of Ns in each strand of the duplexis between about 12 and about 50 (i.e., in the figure above, oligo(N)has between about 12 and about 50 nucleomonomers). In other embodiments,the number of Ns in each strand of the duplex is between about 12 andabout 40; or between about 15 and about 35; or more particularly betweenabout 20 and about 30; or even between about 21 and about 25.

[0010] In one embodiment, X is a sequence of about 4 to about 20nucleomonomers which form a loop, wherein the nucleomonomers areselected from the group consisting of G and A.

[0011] In one embodiment, two of the Ns are unlinked, i.e., there is nophosphodiester bond between the two nucleomonomers. In one embodiment,the unlinked Ns are not in the antisense sequence.

[0012] In one embodiment, the nucleotide sequence of the loop is GAAA.

[0013] In another aspect, the invention pertains to a double-strandedoligonucleotide composition having the structure:

[0014] where (1) N is a nucleomonomer in complementary oligonucleotidestrands of equal length where the sequence of Ns corresponds to a targetgene sequence; and (2) Z is a nucleomonomer in complementaryoligonucleotide strands of between about 2 and about 8 nucleomonomers inlength and where the sequence of Zs optionally corresponds to the targetsequence; and (3) where M is a nucleomonomer in complementaryoligonucleotide strands of between about 2 and about 8 nucleomonomers inlength and where the sequence of Ms optionally corresponds to the targetsequence. Although the sequences of N nucleomonomers should be of thesame length, the sequences of Z and M nucleomonomers may optionally beof the same length.

[0015] In one embodiment, Z and M are nucleomonomers selected from thegroup consisting of C and G.

[0016] In one embodiment, the sequence of Zs or Ms is CC, GG, CG, GC,CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC,CCCG, CGGG, GCCC, GGCC, or CCGG.

[0017] In another aspect, the invention pertains to a double-strandedoligonucleotide composition having the structure:

[0018] where (1) N is a nucleomonomer in complementary oligonucleotidestrands of equal length and where the sequence of Ns corresponds to atarget gene sequence and (2) X is selected from the group consisting ofnothing; 1-20 nucleotides of 5′ overhang; 1-20 nucleotides of 3′overhang.

[0019] In some embodiments, X is a loop structure consisting of fromabout 4 to about 20 nucleomonomers, where the nucleomonomers areselected from the group consisting of G and A.

[0020] In the structure above, M is a nucleomonomer in complementaryoligonucleotide strands of between about 2 and about 8 nucleomonomers inlength which optionally correspond to the target sequence. In oneembodiment, M is a nucleomonomer selected from the group consisting ofcontain C and G.

[0021] In one embodiment, the sequence of M is CC, GG, CG, GC, CCC, GGG,CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG,CGGG, GCCC, GGCC, or CCGG.

[0022] In another aspect, the invention pertains to a double-strandedoligonucleotide composition having the structure:

[0023] where (1) N is a nucleomonomer in complementary oligonucleotidestrands of equal length and which correspond to a target gene sequenceand (2) Y is selected from the group consisting of nothing; 1-20nucleotides of 5′ overhang; 1-20 nucleotides of 3′ overhang; a loopconsisting of a sequence of from about 4 to about 20 nucleomonomers,where the nucleomonomers are all either Gs or A's and (3) where Z is anucleomonomer in complementary oligonucleotide strands of between about2 and about 8 nucleomonomers in length and which comprise a sequencewhich can optionally correspond to the target sequence.

[0024] In one embodiment, Zs are nucleomonomers selected from the groupconsisting of C and G.

[0025] In one embodiment, the sequence of Zs is CC, GG, CG, GC, CCC,GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG,CGGG, GCCC, GGCC, or CCGG.

[0026] In another aspect, the invention pertains to a method ofregulating gene expression in a cell, comprising forming adouble-stranded oligonucleotide composition as described herein andcontacting a cell with the double-stranded duplex, to thereby regulategene expression in a cell.

[0027] In one embodiment, the invention pertains to a method ofincreasing the nuclease resistance of an antisense sequence, comprisingforming a double-stranded oligonucleotide composition as describedherein, such that a double-stranded duplex is formed, wherein thenuclease resistance of the antisense sequence is increased compared to adouble-stranded, unmodified RNA molecule.

[0028] Methods of stabilizing oligonucleotides, particularly antisenseoligonucleotides, by formation of a oligonucleotide compositionscomprising at least 3 different oligonucleotides, are disclosed inco-pending application no. U.S. Ser. No. ______, filed on the same dayas the present application, bearing attorney docket number “SRI-013,”and entitled “Oligonucleotide Compositions with Enhanced Efficiency.”This application and all of its teachings is hereby expresslyincorporated herein by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 shows that the length of double-stranded oligonucleotidesand the presence or absence of overhangs has no effect on function.

[0030]FIG. 1B shows the effect of structural changes on the efficacy ofsiRNAs targeting β-3-Integrin.

[0031]FIG. 2 shows that there is no correlation was observed between thelength of the double-stranded oligonucleotide and the level of PKRinduction for the given sequences.

[0032]FIG. 2B shows effect of β-3-integrin targeted 21-mer and 27-merson PKR expression in HMVEC Cells.

[0033]FIG. 3 shows the effect of 5′ or 3′ modification on activity ofdouble-stranded RNA duplexes.

[0034]FIG. 4 shows the effect of the size of the modifying group onactivity of the double-stranded RNA duplex.

[0035]FIG. 5 shows the results of 2′-O-Me modifications on the activityof double-stranded RNA duplexes.

[0036]FIG. 6 shows the inhibition of p53 by 32- and 37-mer blunt-endsiRNAs.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The instant invention advances the prior art by providingdouble-stranded oligonucleotide compositions for use, both in vitro andin vivo, e.g., therapeutically, and by providing methods of making andusing the double-stranded antisense oligomer compositions.

[0038] Double-Stranded Oligonucleotide Compositions

[0039] Double-stranded oligonucleotides of the invention are capable ofinhibiting the synthesis of a target protein, which is encoded by atarget gene. The target gene can be endogenous or exogenous (e.g.,introduced into a cell by a virus or using recombinant DNA technology)to a cell. As used herein, the term “target gene” includespolynucleotides comprising a region that encodes a polypeptide orpolynucleotide region that regulates replication, transcription,translation, or other process important in expression of the targetprotein; or a polynucleotide comprising a region that encodes the targetpolypeptide and a region that regulates expression of the targetpolypeptide; or non-coding regions such as the 5′ or 3′ UTR or introns.Accordingly, the term “target gene” as used herein may refer to, forexample, an mRNA molecule produced by transcription a gene of interest.Furthermore, the term “correspond,” as in “an oligomer corresponds to atarget gene sequence,” means that the two sequences are complementary orhomologous or bear such other biologically rational relationship to eachother (e.g., based on the sequence of nucleomonomers and theirbase-pairing properties).

[0040] The “target gene” to which an RNA molecule of the invention isdirected may be associated with a pathological condition. For example,the gene may be a pathogen-associated gene, e.g., a viral gene, atumor-associated gene, or an autoimmune disease-associated gene. Thetarget gene may also be a heterologous gene expressed in a recombinantcell or a genetically altered organism. By determining or modulating(e.g., inhibiting) the function of such a gene, valuable information andtherapeutic benefits in medicine, veterinary medicine, and biology maybe obtained.

[0041] The term “oligonucleotide” includes two or more nucleomonomerscovalently coupled to each other by linkages (e.g., phosphodiesters) orsubstitute linkages. In one embodiment, it may be desirable to use asingle-stranded nucleic acid molecule which forms a duplex structure(e.g., as described in more detail below). For example, in oneembodiment, the oligonucleotide can include a nick in either the senseof the antisense sequence.

[0042] The term “antisense” refers to a nucleotide sequence that isinverted relative to its normal orientation for transcription and soexpresses an RNA transcript that is complementary to a target gene mRNAmolecule expressed within the host cell (e.g., it can hybridize to thetarget gene mRNA molecule through Watson-Crick base pairing). Anantisense strand may be constructed in a number of different ways,provided that it is capable of interfering with the expression of atarget gene. For example, the antisense strand can be constructed byinverting the coding region (or a portion thereof) of the target generelative to its normal orientation for transcription to allow thetranscription of its complement, (e.g., RNAs encoded by the antisenseand sense gene may be complementary). Furthermore, the antisenseoligonucleotide strand need not have the same intron or exon pattern asthe target gene, and noncoding segments of the target gene may beequally effective in achieving antisense suppression of target geneexpression as coding segments.

[0043] Accordingly, one aspect of the invention is a method ofinhibiting the activity of a target gene by introducing an RNAi agentinto a cell, such that the dsRNA component of the RNAi agent is targetedto the gene. In one embodiment, an RNA oligonucleotide molecule maycontain at least one nucleomonomer that is a modified nucleotideanalogue. The nucleotide analogues may be located at positions where thetarget-specific activity, e.g., the RNAi mediating activity is notsubstantially effected, e.g., in a region at the 5′-end or the 3′-end ofthe double-stranded molecule, where the overhangs may be stabilized byincorporating modified nucleotide analogues.

[0044] In another aspect, double-stranded RNA molecules known in the artcan be used in the methods of the present invention. Double-stranded RNAmolecules known in the art may also be modified according to theteachings herein in conjunction with such methods, e.g., by usingmodified nucleomonomers. For example, see U.S. Pat. No. 6,506,559; U.S.Pat. No. 2002/0,173,478 A1; U.S. Pat. No. 2002/0,086,356 A1; Shuey, etal., “RNAi: gene-silencing in therapeutic intervention.” Drug Discov.Today 2002 Oct 15;7(20):1040-6; Aoki, et al., “Clin. Exp. Pharmacol.Physiol. 2003 Jan;30(1-2):96-102; Cioca, et al., “RNA interference is afunctional pathway with therapeutic potential in human myeloid leukemiacell lines. Cancer Gene Ther. 2003 Feb;10(2):125-33.

[0045] Further examples of double-stranded RNA molecules include thosedisclosed in the following references: Kawasaki, et al., “Short hairpintype of dsRNAs that are controlled by tRNA(Val) promoter significantlyinduce RNAi-mediated gene silencing in the cytoplasm of human cells.”Nucleic Acids Res. 2003 Jan 15;31(2):700-7; Cottrell, et al., “Silenceof the strands: RNA interference in eukaryotic pathogens.” TrendsMicrobiol. 2003 Jan; 11(1):37-43; Links, “Mammalian RNAi for themasses.” Trends Genet. 2003 Jan;19(1):9-12; Hamada, et al., “Effects onRNA interference in gene expression (RNAi) in cultured mammalian cellsof mismatches and the introduction of chemical modifications at the3′-ends of siRNAs.” Antisense Nucleic Acid Drug Dev. 2002Oct;12(5):301-9; Links, “RNAi and related mechanisms and their potentialuse for therapy.” Curr. Opin. Chem. Biol. 2002 Dec;6(6):829-34;Kawasaki, et al., “Short hairpin type of dsRNAs that are controlled bytRNA(Val) promoter significantly induce RNAi-mediated gene silencing inthe cytoplasm of human cells.” Nucleic Acids Res. 2003 Jan15;31(2):700-7.)

[0046] A nick is two non-linked nucleomonomers in an oligonucleotide. Anick can be included at any point along the sense or antisensenucleotide sequence. In a preferred embodiment, a nick is in the sensesequence. In another preferred embodiment, the nick is at least aboutfour nucleomonomers in from an end of the duplexed region of theoligonucleotide (e.g., is at least about four nucleomonomers away fromthe 5′ or 3′ end of the oligonucleotide or away from a loop structure.For example, in one embodiment, the nick is present in the middle of thesense strand of the duplex molecule (e.g., if the sense sequence of theduplex is 30 nucleomonomers in length, nucleomonomers 14 and 15 or 15and 16 are unlinked). In an embodiment, a nick may optionally be ligatedto form a circular nucleic acid molecule.

[0047] For example, in the structure below, the indicated Unucleomonomer is not bonded to the neighboring nucleomonomer, e.g., by aphosphodiester bond. The 5′ OH of the nick may optionally bephosphorylated to allow enzymatic ligation of the oligonucleotide into acircle.

[0048] As used herein, the term “nucleotide” includes any monomeric unitof DNA or RNA containing a sugar moiety (pentose), a phosphate, and anitrogenous heterocyclic base. The base is usually linked to the sugarmoiety via the glycosidic carbon (at the 1′ carbon of pentose) and thatcombination of base and sugar is called a “nucleoside.” The basecharacterizes the nucleotide with the four customary bases of DNA beingadenine (A), guanine (G), cytosine (C) and thymine (T). Inosine (I) isan example of a synthetic base that can be used to substitute for any ofthe four, naturally-occurring bases (A, C, G, or T). The four RNA basesare A, G, C, and uracil (U). Accordingly, an oligonucleotide may be anucleotide sequence comprising a linear array of nucleotides connectedby phosphodiester bonds between the 3′ and 5′ carbons of adjacentpentoses. Other modified nucleosides/nucleotides are described hereinand may also be used in the oligonucleotides of the invention.

[0049] Oligonucleotides may comprise, for example, oligonucleotides,oligonucleosides, polydeoxyribonucleotides (containing2′-deoxy-D-ribose) or modified forms thereof, e.g., DNA,polyribonucleotides (containing D-ribose or modified forms thereof),RNA, or any other type of polynucleotide which is an N-glycoside orC-glycoside of a purine or pyrimidine base, or modified purine orpyrimidine base. The term oligonucleotide includes compositions in whichadjacent nucleomonomers are linked via phosphorothioate, amide or otherlinkages (e.g., Neilsen, P. E., et al. 1991. Science. 254:1497).Generally, the term “linkage” refers to any physical connection,preferably covalent coupling, between two or more nucleic acidcomponents, e.g., catalyzed by an enzyme such as a ligase.

[0050] In addition to its art-recognized meaning (e.g., a relativelyshort length single or double-stranded sequences of deoxyribonucleotidesor ribonucleotides linked via phosphodiester bonds), the term“oligonucleotide” includes any structure that serves as a scaffold orsupport for the bases of the oligonucleotide, where the scaffold permitsbinding to the target nucleic acid molecule in a sequence-dependentmanner.

[0051] Oligonucleotides of the invention are isolated. The term“isolated” includes nucleic acid molecules which are synthesized (e.g.,chemically, enzymatically, or recombinantly) or are naturally occurringbut separated from other nucleic acid molecules which are present in anatural source of the nucleic acid. Preferably, a naturally occurring“isolated” nucleic acid molecule is free of sequences which naturallyflank the nucleic acid molecule (i.e., sequences located at the 5′ and3′ ends of the nucleic acid molecule) in a nucleic acid molecule in anorganism from which the nucleic acid molecule is derived.

[0052] The term “nucleomonomer” includes a single base covalently linkedto a second moiety. Nucleomonomers include, for example, nucleosides andnucleotides. Nucleomonomers can be linked to form oligonucleotides thatbind to target nucleic acid sequences in a sequence specific manner.

[0053] In one embodiment, modified (non-naturally occurring)nucleomonomers can be used in the oligonucleotides described herein. Forexample, nucleomonomers which are based on bases (purines, pyrimidines,and derivatives and analogs thereof) bound to substituted andunsubstituted cycloalkyl moieties, e.g., cyclohexyl or cyclopentylmoieties, and substituted and unsubstituted heterocyclic moieties, e.g.,6-member morpholino moieties or, preferably, sugar moieties.

[0054] Sugar moieties include natural, unmodified sugars, e.g.,monosaccharides (such as pentoses, e.g., ribose, deoxyribose), modifiedsugars and sugar analogs. Possible modifications of nucleomonomers,particularly of a sugar moiety, include, for example, replacement of oneor more of the hydroxyl groups with a halogen, a heteroatom, analiphatic group, or the functionalization of the hydroxyl group as anether, an amine, a thiol, or the like. One particularly useful group ofmodified nucleomonomers are 2′-O-methyl nucleotides, especially when the2′-O-methyl nucleotides are used as nucleomonomers in the ends of theoligomers. Such 2′O-methyl nucleotides may be referred to as“methylated,” and the corresponding nucleotides may be made fromunmethylated nucleotides followed by alkylation or directly frommethylated nucleotide reagents. Modified nucleomonomers may be used incombination with unmodified nucleomonomers. For example, anoligonucleotide of the invention may contain both methylated andunmethylated nucleomonomers.

[0055] Some exemplary modified nucleomonomers include sugar-orbackbone-modified ribonucleotides. Modified ribonucleotides may containa nonnaturally occurring base (instead of a naturally occurring base)such as uridines or cytidines modified at the 5-position, e.g.,5-(2-amino)propyl uridine and 5-bromo uridine; adenosines and guanosinesmodified at the 8-position, e.g., 8-bromo guanosine; deaza nucleotides,e.g., 7-deaza-adenosine; and N-alkylated nucleotides, e.g., N6-methyladenosine. Also, sugar-modified ribonucleotides may have the 2′-OH groupreplaced by a H, alxoxy (or OR), R or alkyl, halogen, SH, SR, amino(such as NH₂, NHR, NR_(2,)), or CN group, wherein R is lower alkyl,alkenyl, or alkynyl.

[0056] Modified ribonucleotides may also have the phosphoester groupconnecting to adjacent ribonucleotides replaced by a modified group,e.g., of phosphothioate group. More generally, the various nucleotidemodifications may be combined.

[0057] In one embodiment, sense oligomers may have 2′ modifications onthe ends (1 on each end, 2 on each end, 3 on each end, and 4 on eachend, and so on; as well as 1 on one end, 2 on one end, 3 on one end, and4 on one end, and so on; and even unbalanced combinations such as 1 onone end and 2 on the other end, and so on). Likewise, the antisensestrand may have 2′ modifications on the ends (1 on each end, 2 on eachend, 3 on each end, and 4 on each end, and so on; as well as 1 on oneend, 2 on one end, 3 on one end, and 4 on one end, and so on; and evenunbalanced combinations such as 1 on one end and 2 on the other end, andso on). In preferred aspects, such 2′-modifications are in the sense RNAstrand or the sequences other than the antisense strand.

[0058] To further maximize endo- and exonuclease resistance, in additionto the use of 2′ modified nucleomonomers in the ends,inter-nucleomonomer linkages other than phosphodiesters may be used. Forexample, such end blocks may be used alone or in conjunction withphosphothorothioate linkages between the 2′-O-methly linkages. Preferred2′-modified nucleomonomers are 2′-modified C and U bases.

[0059] Although the antisense strand may be substantially identical toat least a portion of the target gene (or genes), at least with respectto the base pairing properties, the sequence need not be perfectlyidentical to be useful, e.g., to inhibit expression of a target gene'sphenotype. Generally, higher homology can be used to compensate for theuse of a shorter antisense gene. In some cases, the antisense strandgenerally will be substantially identical (although in antisenseorientation) to the target gene.

[0060] One particular example of a composition of the invention hasend-blocks on both ends of a sense oligonucleotide and only the 3′ endof an antisense oligonucleotide. Without wishing to be bound by theory,the inventors believe that a 2′-O-modified sense strand works less wellthan unmodified because it is not efficiently unwound. Accordingly,another embodiment of the invention includes duplexes in whichnucleomonomer-nucleomonomer mismatches are present in a sense2′-O-methly strand (and are thought to be easier to unwind).

[0061] Accordingly, for a given first oligonucleotide strand, a numberof complementary second oligonucleotide strands are permitted accordingto the invention. For example, in the following Tables, a targeted and anon-targeted oligonucleotide are illustrated with several possiblecomplementary oligonucleotides. The individual nucleotides may be 2′-OHRNA nucleotides (R) or the corresponding 2′-OMe nucleotides (M), and theoligonucleotides themselves may contain mismatched nucleotides (lowercase letters).

[0062] Targeted Oligonucleotide: First CCCUUCUGUCUUGAACAUGAG (SEQ ID NO:##) Strand: Second CTgATGTTCAAGACAGAAcGG (SEQ ID NO: ##) Strand: (methylMMMMMMMMMMMMMMMMMMMMM groups →) CTgATGTTCAAGACAGAAcGG (SEQ ID NO: ##)RRRRRRRRRRRRRRRRRRRDD CTCAUGUUCAAGACAGAAGGG (SEQ ID NO: ##)RRRRRRMMMMMMMMMRRRRRR CTCAUGUUCAAGACAGAAGGG (SEQ ID NO: ##)MMMMMMRRRRRRRRRMMMMMM CTCAUGUUCAAGACAGAAGGG (SEQ ID NO: ##)RMRMRMRMRMRMRMRMRMRMR

[0063] Non-Targeted Oligonucleotide: First GAGTACAAGTTCTGTCTTCCC (SEQ IDNO: ##) Strand: Second GGcAAGACAGAACTTGTAgTC (SEQ ID NO: ##) Strand:(methyl MMMMMMMMMMMMMMMMMMMMM groups →) GGGAAGACAGAACTTGTACTC (SEQ IDNO: ##) RRRRRRMMMMMMMMMRRRRRR GGGAAGACAGAACTTGTACTC (SEQ ID NO: ##)MMMMMMRRRRRRRRRMMMMMM GGGAAGACAGAACTTGTACTC (SEQ ID NO: ##)RMRMRMRMRMRMRMRMRMRMR

[0064] Another example of further modifications that may be used inconjunction with 2′-O-methyl nucleomonomers are modification of thesugar residues themselves, for example alternating modified andunmodified sugars, particularly in the sense strand.

[0065] In some embodiments, the length of the sense strand can be 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides, with acomplementary duplexed RNA strand, optionally having overhangs.

[0066] As a further example, the use of 2′-O-methyl RNA may beneficiallybe used in circumstances in which it is desirable to minimize cellularstress responses. RNA having 2′-O-methyl nucleomonomers may not berecognized by cellular machinery that is thought to recognize unmodifiedRNA. The use of 2′-O-methylated or partially 2′-O-methylated RNA mayavoid the interferon response to double-stranded nucleic acids, whilemaintaining target RNA inhibition. This RNAi (“stealth RNAi”) is usefulfor avoiding the interferon or other cellular stress responses, both inshort RNAi (e.g., siRNA) sequences that induce the interferon response,and in longer RNAi sequences that may induce the interferon response.

[0067] An especially advantageous use of the present invention is ingene function studies in which multiple RNAi sequences are used.According to present methods known in the art, frequently there is noway of predicting which sequences might induce a stress response,including the interferon response, and in this regard the presentinvention significantly advances the state of the art. For example, ifall of the multiple sequences are partially 2-O-methylated, the stressresponse, including interferon response, may be avoided, and thus avoidconfounding results in which some sequences affect cellular phenotypeindependent of the target gene inhibition. Other chemical modificationsin addition to 2′-O-methylation may also achieve this effect.

[0068] For example, modified sugars include D-ribose, 2′-O-alkyl(including 2′-O-methyl and 2′-O-ethyl), i.e., 2′-alkoxy, 2′-amino,2′-S-alkyl, 2′-halo (including 2′-fluoro), 2′-methoxyethoxy, 2′-allyloxy(—OCH₂CH═CH₂), 2′-propargyl, 2′-propyl, ethynyl, ethenyl, propenyl, andcyano and the like. In one embodiment, the sugar moiety can be a hexoseand incorporated into an oligonucleotide as described (Augustyns, K., etal., Nucl. Acids. Res. 1992. 18:4711). Exemplary nucleomonomers can befound, e.g., in U.S. Pat. No. 5,849,902, incorporated by referenceherein.

[0069] The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.In certain embodiments, a straight chain or branched chain alkyl has 6or fewer carbon atoms in its backbone (e.g., C₁-C₆ for straight chain,C₃-C₆ for branched chain), and more preferably 4 or fewer. Likewise,preferred cycloalkyls have from 3-8 carbon atoms in their ringstructure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₁-C₆ includes alkyl groups containing 1 to 6 carbonatoms.

[0070] Moreover, unless otherwise specified, the term alkyl includesboth “unsubstituted alkyls” and “substituted alkyls,” the latter ofwhich refers to alkyl moieties having substituents replacing a hydrogenon one or more carbons of the hydrocarbon backbone. Such substituentscan include, for example, alkenyl, alkynyl, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “alkylaryl” or an “arylalkyl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl”also includes the side chains of natural and unnatural amino acids. Theterm “n-alkyl” means a straight chain (i.e., unbranched) unsubstitutedalkyl group.

[0071] The term “alkenyl” includes unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double bond. For example, the term“alkenyl” includes straight-chain alkenyl groups (e.g., ethylenyl,propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl,decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic)groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, andcycloalkyl or cycloalkenyl substituted alkenyl groups. In certainembodiments, a straight chain or branched chain alkenyl group has 6 orfewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). Likewise, cycloalkenyl groups may have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₂-C₆ includes alkenyl groupscontaining 2 to 6 carbon atoms.

[0072] Moreover, unless otherwise specified, the term alkenyl includesboth “unsubstituted alkenyls” and “substituted alkenyls,” the latter ofwhich refers to alkenyl moieties having substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents can include, for example, alkyl groups, alkynyl groups,halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

[0073] The term “alkynyl” includes unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but which contain at least one triple bond. For example, the term“alkynyl” includes straight-chain alkynyl groups (e.g., ethynyl,propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl,decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl orcycloalkenyl substituted alkynyl groups. In certain embodiments, astraight chain or branched chain alkynyl group has 6 or fewer carbonatoms in its backbone (e.g., C₂-C₆ for straight chain, C₃-C₆ forbranched chain). The term C₂-C₆ includes alkynyl groups containing 2 to6 carbon atoms.

[0074] Moreover, unless otherwise specified, the term alkynyl includesboth “unsubstituted alkynyls” and “substituted alkynyls,” the latter ofwhich refers to alkynyl moieties having substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents can include, for example, alkyl groups, alkynyl groups,halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

[0075] Unless the number of carbons is otherwise specified, “loweralkyl” as used herein means an alkyl group, as defined above, but havingfrom one to five carbon atoms in its backbone structure. “Lower alkenyl”and “lower alkynyl” have chain lengths of, for example, 2-5 carbonatoms.

[0076] The term “alkoxy” includes substituted and unsubstituted alkyl,alkenyl, and alkynyl groups covalently linked to an oxygen atom.Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy,propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxygroups include halogenated alkoxy groups. The alkoxy groups can besubstituted with groups such as alkenyl, alkynyl, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.Examples of halogen substituted alkoxy groups include, but are notlimited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

[0077] The term “heteroatom” includes atoms of any element other thancarbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfurand phosphorus.

[0078] The term “hydroxy” or “hydroxyl” includes groups with an —OH or—O⁻ (with an appropriate counterion).

[0079] The term “halogen” includes fluorine, bromine, chlorine, iodine,etc. The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

[0080] The term “substituted” includes substituents which can be placedon the moiety and which allow the molecule to perform its intendedfunction. Examples of substituents include alkyl, alkenyl, alkynyl,aryl, (CR′R″)₀₋₃NR′R″, (CR′R″)₀₋₃CN, NO₂, halogen,(CR′R″)_(O-3)C(halogen)₃, (CR′R″)₀₋₃CH(halogen)₂,(CR′R″)₀₋₃CH₂(halogen), (CR′R″)₀₋₃CONR′R″, (CR′R″)₀₋₃S(O)₁₋₂NR′R″,(CR′R″)₀₋₃CHO, (CR′R″)₀₋₃O(CR′R″)₀₋₃H, (CR′R″)₀₋₃S(O)₀₋₂R′,(CR′R″)₀₋₃O(CR′R″)₀₋₃H, (CR′R″)₀₋₃COR′, (CR′R″)₀₋₃CO₂R′, or(CR′R″)₀₋₃OR′ groups; wherein each R′ and R″ are each independentlyhydrogen, a C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, or aryl group, orR′ and R″ taken together are a benzylidene group or a —(CH₂)₂O (CH₂)₂-group.

[0081] The term “amine” or “amino” includes compounds or moieties inwhich a nitrogen atom is covalently bonded to at least one carbon orheteroatom. The term “alkyl amino” includes groups and compounds whereinthe nitrogen is bound to at least one additional alkyl group. The term“dialkyl amino” includes groups wherein the nitrogen atom is bound to atleast two additional alkyl groups.

[0082] The term “ether” includes compounds or moieties which contain anoxygen bonded to two different carbon atoms or heteroatoms. For example,the term includes “alkoxyalkyl,” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

[0083] The term “base” includes the known purine and pyrimidineheterocyclic bases, deazapurines, and analogs (including heterocyclicsubstituted analogs, e.g., aminoethyoxy phenoxazine), derivatives (e.g.,1-alkyl-, 1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) andtautomers thereof. Examples of purines include adenine, guanine,inosine, diaminopurine, and xanthine and analogs (e.g.,8-oxo-N⁶-methyladenine or 7-diazaxanthine) and derivatives thereof.Pyrimidines include, for example, thymine, uracil, and cytosine, andtheir analogs (e.g., 5-methylcytosine, 5-methyluracil,5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and 4,4-ethanocytosine).Other examples of suitable bases include non-purinyl and non-pyrimidinylbases such as 2-aminopyridine and triazines.

[0084] In a preferred embodiment, the nucleomonomers of anoligonucleotide of the invention are RNA nucleotides. In anotherpreferred embodiment, the nucleomonomers of an oligonucleotide of theinvention are modified RNA nucleotides.

[0085] The term “nucleoside” includes bases which are covalentlyattached to a sugar moiety, preferably ribose or deoxyribose. Examplesof preferred nucleosides include ribonucleosides anddeoxyribonucleosides. Nucleosides also include bases linked to aminoacids or amino acid analogs which may comprise free carboxyl groups,free amino groups, or protecting groups. Suitable protecting groups arewell known in the art (see P. G. M. Wuts and T. W. Greene, “ProtectiveGroups in Organic Synthesis”, 2^(nd) Ed., Wiley-Interscience, New York,1999).

[0086] The term “nucleotide” includes nucleosides which further comprisea phosphate group or a phosphate analog.

[0087] As used herein, the term “linkage” includes a naturallyoccurring, unmodified phosphodiester moiety (—O—(PO₂ ⁻)—O—) thatcovalently couples adjacent nucleomonomers. As used herein, the term“substitute linkage” includes any analog or derivative of the nativephosphodiester group that covalently couples adjacent nucleomonomers.Substitute linkages include phosphodiester analogs, e.g.,phosphorothioate, phosphorodithioate, and P-ethyoxyphosphodiester,P-ethoxyphosphodiester, P-alkyloxyphosphotriester, methylphosphonate,and nonphosphorus containing linkages, e.g., acetals and amides. Suchsubstitute linkages are known in the art (e.g., Bjergarde et al. 1991.Nucleic Acids Res. 19:5843; Caruthers et al. 1991. NucleosidesNucleotides. 10:47).

[0088] In certain embodiments, oligonucleotides of the inventioncomprise 3′ and 5′ termini (except for circular oligonucleotides). Inone embodiment, the 3′ and 5′ termini of an oligonucleotide can besubstantially protected from nucleases e.g., by modifying the 3′ or 5′linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For example,oligonucleotides can be made resistant by the inclusion of a “blockinggroup.” The term “blocking group” as used herein refers to substituents(e.g., other than OH groups) that can be attached to oligonucleotides ornucleomonomers, either as protecting groups or coupling groups forsynthesis (e.g., FITC, propyl (CH₂-CH₂-CH₃), phosphate (PO₃ ²⁻),hydrogen phosphonate, or phosphoramidite). “Blocking groups” alsoinclude “end blocking groups” or “exonuclease blocking groups” whichprotect the 5′ and 3′ termini of the oligonucleotide, including modifiednucleotides and non-nucleotide exonuclease resistant structures.

[0089] Exemplary end-blocking groups include cap structures (e.g., a7-methylguanosine cap), inverted nucleomonomers, e.g., with 3′-3′ or5′-5′ end inversions (see, e.g., Ortiagao et al. 1992. Antisense Res.Dev. 2:129), methylphosphonate, phosphoramidite, non-nucleotide groups(e.g., non-nucleotide linkers, amino linkers, conjugates) and the like.The 3′ terminal nucleomonomer can comprise a modified sugar moiety. The3′ terminal nucleomonomer comprises a 3′-O that can optionally besubstituted by a blocking group that prevents 3′-exonuclease degradationof the oligonucleotide. For example, the 3′-hydroxyl can be esterifiedto a nucleotide through a 3′→3′ internucleotide linkage. For example,the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, andpreferably, ethoxy. Optionally, the 3′→3′ linked nucleotide at the 3′terminus can be linked by a substitute linkage. To reduce nucleasedegradation, the 5′ most 3′→5′ linkage can be a modified linkage, e.g.,a phosphorothioate or a P-alkyloxyphosphotriester linkage. Preferably,the two 5′ most 3′→5′ linkages are modified linkages. Optionally, the 5′terminal hydroxy moiety can be esterified with a phosphorus containingmoiety, e.g., phosphate, phosphorothioate, or P-ethoxyphosphate.

[0090] In one embodiment, the sense strand of an oligonucleotidecomprises a 5′ group that allows for RNAi activity but which renders thesense strand inactive in terms of gene targeting. Preferably, such a 5′modifying group is a phosphate group or a group larger than a phosphategroup

[0091] In another embodiment, the antisense strand of an oligonucleotidecomprises a 5′ phosphate group.

[0092] In one embodiment, the oligonucleotides included in thecomposition are high affinity oligonucleotides. The term “high affinity”as used herein includes oligonucleotides that have a Tm (meltingtemperature) of or greater than about 60° C., greater than about 65° C.,greater than about 70° C., greater than about 75° C., greater than about80° C. or greater than about 85° C. The Tm is the midpoint of thetemperature range over which the oligonucleotide separates from thetarget nucleotide sequence. At this temperature, 50% helical(hybridized) versus coil (unhybridized) forms are present. Tm ismeasured by using the UV spectrum to determine the formation andbreakdown (melting) of hybridization. Base stacking occurs duringhybridization, which leads to a reduction in UV absorption. Tm dependsboth on GC content of the two nucleic acid molecules and on the degreeof sequence complementarity. Tm can be determined using techniques thatare known in the art (see for example, Monia et al. 1993. J Biol. Chem.268:145; Chiang et al. 1991. J Biol. Chem. 266:18162; Gagnor et al.1987. Nucleic Acids Res. 15:10419; Monia et al. 1996. Proc. Natl. Acad.Sci. 93:15481; Publisis and Tinoco. 1989. Methods in Enzymology 180:304;Thuong et al. 1987. Proc. Natl. Acad. Sci. USA 84:5129).

[0093] In one embodiment, an oligonucleotide can include an agent whichincreases the affinity of the oligonucleotide for its target sequence.The term “affinity enhancing agent” includes agents that increase theaffinity of an oligonucleotide for its target. Such agents include,e.g., intercalating agents and high affinity nucleomonomers.Intercalating agents interact strongly and nonspecifically with nucleicacids. Intercalating agents serve to stabilize RNA-DNA duplexes and thusincrease the affinity of the oligonucleotides for their targets.Intercalating agents are most commonly linked to the 3′ or 5′ end ofoligonucleotides. Examples of intercalating agents include acridine,chlorambucil, benzopyridoquinoxaline, benzopyridoindole,benzophenanthridine, and phenazinium. The agents may also impart othercharacteristics to the oligonucleotide, for example, increasingresistance to endonucleases and exonucleases.

[0094] In one embodiment, a high affinity nucleomonomer is incorporatedinto an oligonucleotide. The language “high affinity nucleomonomer” asused herein includes modified bases or base analogs that bind to acomplementary base in a target nucleic acid molecule with higheraffinity than an unmodified base, for example, by having moreenergetically favorable interactions with the complementary base, e.g.,by forming more hydrogen bonds with the complementary base. For example,high affinity nucleomonomer analogs such as aminoethyoxy phenoxazine(also referred to as a G clamp), which forms four hydrogen bonds withguanine are included in the term “high affinity nucleomonomer.” A highaffinity nucleomonomer is illustrated below (see, e.g., Flanagan, etal., 1999. Proc. Natl. Acad. Sci. 96:3513).

[0095] Other exemplary high affinity nucleomonomers are known in the artand include 7-alkenyl, 7-alkynyl, 7-heteroaromatic-, or7-alkynyl-heteroaromatic-substituted bases or the like which can besubstituted for adenosine or guanosine in oligonucleotides (see, e.g.,U.S. Pat. No. 5,594,121). Also, 7-substituted deazapurines have beenfound to impart enhanced binding properties to oligonucleotides, i.e.,by allowing them to bind with higher affinity to complementary targetnucleic acid molecules as compared to unmodified oligonucleotides. Highaffinity nucleomonomers can be incorporated into the oligonucleotides ofthe instant invention using standard techniques.

[0096] In another embodiment, an agent that increases the affinity of anoligonucleotide for its target comprises an intercalating agent. As usedherein, the language “intercalating agent” includes agents which canbind to a DNA double helix. When covalently attached to anoligonucleotide of the invention, an intercalating agent enhances thebinding of the oligonucleotide to its complementary genomic DNA targetsequence. The intercalating agent may also increase resistance toendonucleases and exonucleases.

[0097] Exemplary intercalating agents are taught by Helene and Thuong(1989. Genome 31:413), and include e.g., acridine derivatives (Lacosteet al. 1997. Nucleic Acids Research. 25:1991; Kukreti et al. 1997.Nucleic Acids Research. 25:4264); quinoline derivatives (Wilson et al.1993. Biochemistry 32:10614); benzo[f]quino[3,4-b]quioxaline derivatives(Marchand et al. 1996. Biochemistry. 35:5022; Escude et al. 1998. Proc.Natl. Acad. Sci. 95:3591).

[0098] Intercalating agents can be incorporated into an oligonucleotideusing any convenient linkage. For example, acridine or psoralen can belinked to the oligonucleotide through any available —OH or —SH group,e.g., at the terminal 5′ position of the oligonucleotide, the 2′positions of sugar moieties, or an OH, NH₂, COOH, or SH incorporatedinto the 5-position of pyrimidines using standard methods.

[0099] In one embodiment, when included in an RNase H activatingantisense nucleotide sequence, an agent that increases the affinity ofan oligonucleotide for its target is not positioned adjacent to an RNaseactivating region of the oligonucleotide, e.g., is positioned adjacentto a non-RNase activating region. Preferably, the agent that increasesthe affinity of an oligonucleotide for its target is placed at adistance as far as possible from the RNase activating domain of thechimeric antisense sequence such that the specificity of the chimericantisense sequence is not altered when compared with the specificity ofa chimeric antisense sequence which lacks the intercalating compound. Inone embodiment, this can be accomplished by positioning the agentadjacent to a non-RNase activating region. The specificity of theoligonucleotide can be tested by demonstrating that transcription of anon-target sequence, preferably a non-target sequence which isstructurally similar to the target (e.g., has some sequence homology oridentity with the target sequence but which is not identical in sequenceto the target), is not inhibited to a greater degree by anoligonucleotide comprising an affinity enhancing agent than by anoligonucleotide directed against the same target that does not comprisean affinity enhancing agent.

[0100] The double-stranded oligonucleotides of the invention may beformed by a single, self-complementary nucleic acid strand or twoseparate complementary nucleic acid strands. Duplex formation can occureither inside or outside the cell containing the target gene.

[0101] As used herein, the term “double-stranded” includes one or morenucleic acid molecules comprising a region of the molecule in which atleast a portion of the nucleomonomers are complementary and hydrogenbond to form a duplex.

[0102] As used herein, the term “duplex” includes the region of thedouble-stranded nucleic acid molecule(s) that is (are) hydrogen bondedto a complementary sequence.

[0103] The double-stranded oligonucleotides of the invention comprise anucleotide sequence that is sense to a target gene and a complementarysequence that is antisense to the target gene. The sense and antisensenucleotide sequences correspond to the target gene sequence, e.g., areidentical or are sufficiently identical to effect target gene inhibition(e.g., are about at least about 98%, 96% identical, 94%, 90% identical,85% identical, or 80% identical) to the target gene sequence.

[0104] When comprised of two separate complementary nucleic acidmolecules, the individual nucleic acid molecules can be of differentlengths.

[0105] In one embodiment, a double-stranded oligonucleotide of theinvention is double-stranded over its entire length, i.e., with nooverhanging single-stranded sequence at either end of the molecule,i.e., is blunt-ended. In another embodiment, a double-strandedoligonucleotide of the invention is not double-stranded over its entirelength. For instance, when two separate nucleic acid molecules are used,one of the molecules, e.g., the first molecule comprising an antisensesequence can be longer than the second molecule hybridizing thereto(leaving a portion of the molecule single-stranded). Likewise, when asingle nucleic acid molecule is used a portion of the molecule at eitherend can remain single-stranded.

[0106] In one embodiment, a double-stranded oligonucleotide of theinvention is double-stranded over at least about 70% of the length ofthe oligonucleotide. In another embodiment, a double-strandedoligonucleotide of the invention is double-stranded over at least about80% of the length of the oligonucleotide. In another embodiment, adouble-stranded oligonucleotide of the invention is double-stranded overat least about 90%-95% of the length of the oligonucleotide. In anotherembodiment, a double-stranded oligonucleotide of the invention isdouble-stranded over at least about 96%-98% of the length of theoligonucleotide.

[0107] In one embodiment, the double-stranded duplex constructs of theinvention can be further stabilized against nucleases by forming loopstructures at the 5′ or 3′ end of the sense or antisense strand of theconstruct. For example, the construct can take the form:

[0108] where the Ns are nucleomonomers in complementary oligonucleotidestrands (i.e., the top N strand is complementary to the bottom N strand)of equal length (e.g., between about 12 and about 40 nucleotides inlength) and X and Y are each independently selected from a groupconsisting of nothing (i.e., the construct is a blunt ended constructwith no loops and no overhang); from about 1 to about 20 nucleotides of5′ overhang; from about 1 to about 20 nucleotides of 3′ overhang; a GAAAloop (tetra-loop); and a loop consisting from about 4 to about 20nucleomonomers (where the nucleomonomers are all either Gs or A's).

[0109] The sequence of Ns corresponds to the target gene sequence (e.g.,is homologous or identical to a nucleotide sequence that is sense orantisense to the target gene sequence), while the nucleotide sequence ofthe loop structure does not correspond to the target gene sequence.

[0110] For example, such loops can comprise all Gs and A's and be fromabout 4 to about 20 nucleotides in length. In one embodiment, such aloop can be a tetra-loop having a sequence GAAA:

[0111] In one embodiment, the number of Ns is about 27.

[0112] In embodiments in which loops are at one or both ends of theconstruct, the oligonucleotide can be divided by having a “nick” whichis two non-linked nucleomonomers at any point along the sense orantisense strand, but preferably along the sense strand. Preferably, thenick is at least four bases from the nearest end of the duplexed region(to provide enough thermodynamic stability).

[0113] In another embodiment, a construct of the invention can take theform:

[0114] where the Ns are complementary nucleomonomers in oligonucleotidestrands of equal length (e.g., between 12-40 nucleomonomers in length);Zs are nucleomonomers in complementary oligonucleotide strands ofbetween about 2 and about 8 nucleomonomers in length and which comprisea sequence which can optionally correspond to the target sequence; andwhere Ms are nucleomonomers in complementary oligonucleotide strands ofbetween about 2 and about 8 nucleomonomers in length and which canoptionally correspond to the target sequence.

[0115] Preferably, the Zs and Ms are nucleomonomers selected from thegroup consisting of Cs and Gs to make the end of the duplex morethermodynamically stable. Ends of duplexes can become single strandedtransiently, and since duplex RNA is more stable than single-strandedRNA, the enhanced stability of the duplex on the ends will result inhigher nuclease stability.

[0116] A preferred sequence for Z or M in the antisense strand is from2-8 nucleomonomers in length or preferably from 3-4 nucleomonomers inlength, e.g., (from 5′ to 3′) CC, GG, CG, GC, CCC, GGG, CGG, GCC, GCG,CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC,or CCGG. The complementary strand would have the correspondingcomplementary sequence.

[0117] In still another embodiment, a construct of the invention has theform:

[0118] where Ns are nucleomonomers in complementary oligonucleotidestrands (i.e., the top N strand is complementary to the bottom N strand)of equal length (e.g., from between about 12 to about 40 nucleomonomersin length) and X is selected from the group consisting of nothing (i.e.,leaving blunt ends with no loop or overhang); 1-20 nucleotides of 5′overhang; 1-20 nucleotides of 3′ overhang; a GAAA loop (tetra-loop); anda loop consisting of from about 4 to about 20 nucleomonomers (where thenucleomonomers are all either Gs or A's) and where Ms are nucleomonomersin complementary oligonucleotide strands of between about 2 and about 8nucleomonomers in length (which can optionally correspond to the targetsequence). Preferably, Ms are nucleomonomers selected from the groupconsisting of contain Cs and Gs.

[0119] A preferred sequence for M in the antisense strand is from 2-8nucleomonomers in length or preferably from 3-4 nucleomonomers inlength, e.g., (from 5′ to 3′) CC, GG, CG, GC, CCC, GGG, CGG, GCC, GCG,CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC,or CCGG and the corresponding complement on the opposite strand.

[0120] In another embodiment, the construct can take the form:

[0121] where Ns are nucleomonomers in complementary oligonucleotidestrands of equal length (e.g., from between about 12 to about 40nucleomonomers in length) and Y is selected from the group consisting ofnothing (i.e., leaving blunt ends with no loop or overhang; 1-20nucleotides of 5′ overhang; 1-20 nucleotides of 3′ overhang; a GAAA loop(tetra-loop); and a loop consisting of a sequence of from about 4 toabout 20 nucleomonomers (where the nucleomonomers are all either Gs orA's) and where Zs are nucleomonomers in complementary oligonucleotidestrands of between about 2 and about 8 nucleomonomers in length andwhich comprise a sequence which can optionally correspond to the targetsequence. Preferably, the Zs are nucleomonomers selected from the groupconsisting of Cs and Gs to make the end of the duplex more stable.

[0122] A preferred sequence for Z in the antisense strand is from 2-8nucleomonomers in length or preferably from 3-4 nucleomonomers inlength, e.g., (from 5′ to 3′) CC, GG, CG, GC, CCC, GGG, CGG, GCC, GCG,CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC orCCGG (and the corresponding complement on the opposite strand). Forexample, in the following structure, GGCC on the end (and itscomplement) confers additional stability:

[0123] The invention also relates to a double-stranded oligonucleotidecomposition having the following structure:

[0124] wherein (1) oligoA is an oligonucleotide of a number ofnucleomonomers; (2) oligoB is an oligonucleotide that has the samenumber of nucleomonomers as oligoA and that is complementary to oligoA;(3)either oligoA or oligoB corresponds to a target gene sequence.

[0125] In this structure, X may be selected from (a) nothing; (b) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 5′ end of oligoA and constituting a 5′ overhang; (c) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 3′ end of oligoB and constituting a 3′ overhang; (d) and anoligonucleotide of about 4 to about 20 nucleomonomers covalently bondedto the 3′ end of oligoB and the 5′ end of oligoA and constituting a loopstructure, where the nucleomonomers are selected from the groupconsisting of G and A.

[0126] Similarly, Y may be selected from (a) nothing; (b) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 5′ end of oligoB and constituting a 5′ overhang; (c) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 3′ end of oligoA and constituting a 3′ overhang; (d) and anoligonucleotide of about 4 to about 20 nucleomonomers covalently bondedto the 3′ end of oligoA and the 5′ end of oligoB and constituting a loopstructure, where the nucleomonomers are selected from the groupconsisting of G and A.

[0127] Similarly, the invention includes a double-strandedoligonucleotide composition having the structure:

[0128] wherein (1) oligoA is 5′-(N)₁₅₋40-(M)₂₋₈-3′ and oligoB is5′-(N)₁₅₋40-(M)₂₋₈-3′, wherein each of N and M is independently anucleomonomer; (2) both of the sequences of Ns are complementaryoligonucleotide strands of equal length having between about 15 and 40nucleomonomers; (3) at least one of the sequences of Ns, optionally withsome or all of the flanking Ms, corresponds to a target gene sequence.Both of the sequences of Ms are complementary oligonucleotide strands ofbetween about 2 and about 8 nucleomonomers in length. The two M strandsare optionally of the same length.

[0129] The group X indicated by the curved line is selected from (a)nothing; (b) an oligonucleotide of about 1 to about 20 nucleotidescovalently bonded to the 5′ end of oligoA and constituting a 5′overhang; (c) an oligonucleotide of about 1 to about 20 nucleotidescovalently bonded to the 3′ end of oligoB and constituting a 3′overhang; (d) and an oligonucleotide of about 4 to about 20nucleomonomers covalently bonded to the 3′ end of oligoB and the 5′ endof oligoA and constituting a loop structure, where the nucleomonomersare selected from the group consisting of G and A.

[0130] Likewise, the invention pertains to a double-strandedoligonucleotide composition having the structure:

[0131] wherein (1) oligoA is 5′-(Z)₂₋₈-(N)₁₂₋₄₀-3′ and oligoB is5′-(Z)₂₋₈-(N)₁₂₋₄₀-3′, wherein each of N and Z is independently anucleomonomer; (2) both of the sequences of Ns are complementaryoligonucleotide strands of equal length having between about 12 and 40nucleomonomers; (3) at least one of the sequences of Ns, optionally withsome or all of the flanking Zs, corresponds to a target gene sequence.Both of the sequences of Zs are complementary oligonucleotide strands ofbetween about 2 and about 8 nucleomonomers in length. The two Z strandsare optionally of the same length.

[0132] Here, Y is selected from (a) nothing; (b) an oligonucleotide ofabout 1 to about 20 nucleotides covalently bonded to the 5′ end ofoligoB and constituting a 5′ overhang; (c) an oligonucleotide of about 1to about 20 nucleotides covalently bonded to the 3′ end of oligoA andconstituting a 3′ overhang; (d) and an oligonucleotide of about 4 toabout 20 nucleomonomers covalently bonded to the 3′ end of oligoA andthe 5′ end of oligoB and constituting a loop structure, where thenucleomonomers are selected from the group consisting of G and A.

[0133] In one embodiment, the double-stranded duplex of anoligonucleotide of the invention is from between about 12 to about 50nucleomonomers in length, i.e., the number of nucleotides of thedouble-stranded oligonucleotide which hybridize to the complementarysequence of the double-stranded oligonucleotide to form thedouble-stranded duplex structure is from about 12 to about 50nuclemonomers in length. In another embodiment, the double-strandedduplex of an oligonucleotide of the invention is from between about 12to about 40 nucleomonomers in length.

[0134] In one embodiment, the double-stranded duplex of anoligonucleotide of the invention is at least about 25 nucleomonomers inlength. In one embodiment, the double-stranded duplex is greater thanabout 25 nucleomonomers in length. In one embodiment, a double-strandedduplex is at least about 26, 27, 28, 29, 30, at least about 40, at leastabout 50, or at least about 60, at least about 70, at least about 80, orat least about 90 nucleomonomers in length. In another embodiment, thedouble-stranded duplex is less than about 25 nucleomonomers in length.In one embodiment, a double-stranded duplex is at least about 10, atleast about 15, at least about 20, at least about 22, at least about 23or at least about 24 nucleomonomers in length.

[0135] In one embodiment, the number of Ns in each strand of the duplexis about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or27. In another embodiment, the number of Ns in each strand of the duplexis about 30, 35, 40, 45, or 50. In one embodiment, the number of Ns ineach strand of the duplex is about 19. In a preferred embodiment, thenumber of Ns in each strand of the duplex is about 27. In anotherembodiment, the number of Ns in each strand of the duplex is about 27(e.g., is 26, 27, or 28). In another embodiment, the number of Ns ineach strand of the duplex is 27.

[0136] In one embodiment, an individual nucleic acid molecule of adouble-stranded oligonucleotide of the invention is at least about 25nucleomonomers in length. For example, when the double-strandedoligonucleotide of the invention is comprised of one nucleic acidmolecule, that individual molecule is at least about 25 nucleomonomersin length or when the double-stranded oligonucleotide of the inventionis comprised of two separate nucleic acid molecules, the length of atleast one of the individual nucleic acid molecules is at least about 25nucleomonomers in length.

[0137] A variety of nucleotides of different lengths may be used. In oneembodiment, an individual nucleic acid molecule comprising adouble-stranded oligonucleotide of the invention is greater than about25 nucleomonomers in length. In one embodiment, an individual nucleicacid molecule comprising a double-stranded oligonucleotide of theinvention is at least about 26, 27, 28, 29, 30, at least about 40, atleast about 50, or at least about 60, at least about 70, at least about80, or at least about 90 nucleomonomers in length. In anotherembodiment, an individual nucleic acid molecule comprising adouble-stranded oligonucleotide of the invention is less than about 25nucleomonomers in length. In one embodiment, an individual nucleic acidmolecule comprising a double-stranded oligonucleotide of the inventionis at least about 10, at least about 15, at least about 20, at leastabout 22, at least about 23 or at least about 24 nucleomonomers inlength.

[0138] The double-stranded molecules of the invention comprise a firstnucleotide sequence which is antisense to at least part of the targetgene and a second nucleotide sequence which is complementary to thefirst nucleotide sequence; i. e., is sense to at least part of thetarget gene. In one embodiment, the second nucleotide sequence of thedouble-stranded molecule comprises a nucleotide sequence which is atleast about 100% complementary to the antisense molecule.

[0139] In another embodiment, the second nucleotide sequence of thedouble-stranded molecule comprises a nucleotide sequence which is atleast about 95% complementary to the antisense molecule. In anotherembodiment, the second nucleotide sequence of the double-strandedmolecule comprises a nucleotide sequence which is at least about 90%complementary to the antisense molecule. In another embodiment, thesecond nucleotide sequence of the double-stranded molecule comprises anucleotide sequence which is at least about 80% complementary to theantisense molecule. In another embodiment, the second nucleotidesequence of the double-stranded molecule comprises a nucleotide sequencewhich is at least about 60% complementary to the antisense molecule. Inanother embodiment, the second nucleotide sequence of thedouble-stranded molecule comprises a nucleotide sequence which is atleast about 100% complementary to the antisense molecule.

[0140] To determine the percent identity of two nucleic acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-identical sequencescan be disregarded for comparison purposes). When a position in thefirst sequence is occupied by the same nucleotide as the correspondingposition in the second sequence, then the molecules are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The percent complementarity can be determined analogously; when aposition in one sequence occupied by a nucleotide that is complementaryto the nucleotide in the other sequence, then the molecules arecomplementary at that position.

[0141] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twonucleotide sequences is determined using e.g., the GAP program in theGCG software package, using a NWSgapdna. CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity between two nucleotidesequences is determined using the algorithm of E. Meyers and W. Miller(Comput. Appl. Biosci. 4:11-17 (1988)) which has been incorporated intothe ALIGN program (version 2.0), using a PAM120 weight residue table, agap length penalty of 12 and a gap penalty of 4.

[0142] The nucleic acid sequences of the present invention can furtherbe used as a “query sequence” to perform alignments against sequences inpublic databases. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. See, e.g.,the NIH website.

[0143] In yet another embodiment, a first antisense sequence of thedouble-stranded molecule hybridizes to its complementary second sequenceof the double-stranded molecule under stringent hybridizationconditions. As used herein, the term “hybridizes under stringentconditions” is intended to describe conditions for hybridization andwashing under which nucleotide sequences at least 60% complementary toeach other typically remain hybridized to each other. Preferably, theconditions are such that sequences at least about 70%, more preferablyat least about 80%, even more preferably at least about 85% or 90%complementary to each other typically remain hybridized to each other.

[0144] Such stringent conditions are known to those skilled in the artand can be found in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., morepreferably at 60° C., and even more preferably at 65° C. Rangesintermediate to the above-recited values, e.g., at 60-65° C. or at55-60° C. are also intended to be encompassed by the present invention.Alternatively, formamide can be included in the hybridization solution,using methods and conditions also known in the art.

[0145] One of the sequences (or molecules) of the double-strandedoligonucleotide of the invention is antisense to the target gene. Asused herein, the term “antisense sequence” includes nucleotide sequenceswhich bind to the “sense” strand of the nucleotide sequence of thetarget gene (e.g., polynucleotides such as DNA, mRNA (includingpre-mRNA)) molecules. When the antisense sequences of the invention bindto nucleic acid molecules, they can bind to any region of a nucleic acidmolecule, including e.g., introns, exons, 5′, or 3′ untranslatedregions. Antisense sequences that work by binding to a target andactivating RNase H preferably bind within an intron, an exon, the 5′untranslated region, or the 3′ untranslated region of a nucleic acidtarget molecule.

[0146] Preferably, the oligonucleotide compositions of the invention donot activate the interferon pathway, e.g., as evidenced by the lack ofinduction of the double-stranded RNA, interferon-inducible proteinkinase, PKR.

[0147] In one embodiment, modifications are made to a double-strandedRNA molecule which would normally activate the interferon pathway suchthat the interferon pathway is not activated. For example, theinterferon pathway is activated by double-stranded unmodified RNA. Thecellular recognition of double-stranded RNA is highly specific andmodifying one or woth of the strands of a double-stranded duplex enablethe double-stranded RNA molecule to evade the double-stranded RNArecognition machinery of the cell but would still allow for theactivation of the RNAi pathway.

[0148] The ability of a double-stranded oligonucleotide to activateinterferon could be assessed by testing for expression of thedouble-stranded RNA, Interferon-Inducible Protein Kinase, PKR usingtechniques known in the art and also testing for the ability of thedouble-stranded molecule to effect target gene inhibition. Accordingly,in one embodiment, the invention provides a method of testing for theability of a double-stranded RNA molecule to induce interferon bytesting for the ability of the oligonucleotide to activate PKR.Compositions that do not activate PKR (i.e., do not activate theinterferon pathway) are then selected for use to inhibit genetranscription in cells, e.g., in therapeutics or functional genomics.

[0149] Without being limited to any particular mechanism of action, anantisense sequence used in a double-stranded oligonucleotide compositionof the invention that can specifically hybridize with a nucleotidesequence within the target gene (i.e., can be complementary to anucleotide sequence within the target gene) may achieve its affectsbased on, e.g.,: (1) binding to target mRNA and stericly blocking theribosome complex from translating the mRNA; (2) binding to target mRNAand triggering mRNA cleavage by RNase H; (3) binding to double-strandedDNA in the nucleus and forming a triple helix; (4) hybridizing to openDNA loops created by RNA polymerase; (5) interfering with mRNA splicing;(6) interfering with transport of mRNA from the nucleus to thecytoplasm; or (7) interfering with translation through inhibition of thebinding of initiation factors or assembly of ribosomal subunits (i.e.,at the start codon).

[0150] In one embodiment, an antisense sequence of the double-strandedoligonucleotides of the invention is complementary to a target nucleicacid sequence over at least about 80% of the length of the antisensesequence. In another embodiment, the antisense sequence of thedouble-stranded oligonucleotide of the invention is complementary to atarget nucleic acid sequence over at least about 90-95% of the length ofthe antisense sequence. In another embodiment, the antisense sequence ofthe double-stranded oligonucleotide of the invention is complementary toa target nucleic acid sequence over the entire length of the antisensesequence.

[0151] In yet another embodiment, an antisense sequence of thedouble-stranded oligonucleotide hybridizes to at least a portion of thetarget gene under stringent hybridization conditions.

[0152] In one embodiment, antisense sequences of the invention aresubstantially complementary to a target nucleic acid sequence. In oneembodiment, an antisense RNA molecule comprises a nucleotide sequencewhich is at least about 100% complementary to a portion of the targetgene. In another embodiment, an antisense RNA molecule comprises anucleotide sequence which is at least about 90% complementary to aportion of the target gene. In another embodiment, an antisense RNAmolecule comprises a nucleotide sequence which is at least about 80%complementary to a portion of the target gene. In another embodiment, anantisense RNA molecule comprises a nucleotide sequence which is at leastabout 60% complementary to a portion of the target gene. In anotherembodiment, an antisense RNA molecule comprises a nucleotide sequencewhich is at least about 100% complementary to a portion of the targetgene. Preferably, no loops greater than about 8 nucleotides are formedby areas of non-complementarity between the oligonucleotide and thetarget.

[0153] In one embodiment, an antisense nucleotide sequence of theinvention is complementary to a target nucleic acid sequence over atleast about 80% of the length of the antisense sequence. In anotherembodiment, an antisense sequence of the invention is complementary to atarget nucleic acid sequence over at least about 90-95% of the length ofthe antisense sequence. In another embodiment, an antisense sequence ofthe invention is complementary to a target nucleic acid sequence overthe entire length of the antisense sequence.

[0154] The antisense sequences used in an oligonucleotide composition ofthe invention may be of any type, e.g., including morpholinooligonucleotides, RNase H activating oligonucleotides, or ribozymes.

[0155] In one embodiment, a double-stranded oligonucleotide of theinvention can comprise (i.e., be a duplex of) one nucleic acid moleculewhich is DNA and one nucleic acid molecule which is RNA.

[0156] Antisense sequences of the invention can be “chimericoligonucleotides” which comprise an RNA-like and a DNA-like region. Thelanguage “RNase H activating region” includes a region of anoligonucleotide, e.g., a chimeric oligonucleotide, that is capable ofrecruiting RNase H to cleave the target RNA strand to which theoligonucleotide binds. Typically, the RNase activating region contains aminimal core (of at least about 3-5, typically between about 3-12, moretypically, between about 5-12, and more preferably between about 5-10contiguous nucleomonomers) of DNA or DNA-like nucleomonomers. (See,e.g., U.S. Pat. No. 5,849,902). Preferably, the RNase H activatingregion comprises about nine contiguous deoxyribose containingnucleomonomers.

[0157] In one embodiment, the contiguous nucleomonomers are linked by asubstitute linkage, e.g., a phosphorothioate linkage. In one embodiment,an antisense sequence of the invention is unstable, i.e., is degraded ina cell, in the absence of the second strand (or self complementarysequence) which forms a double-stranded oligonucleotide of theinvention. For example, in one embodiment, a chimeric antisense sequencecomprises unmodified DNA nucleomonomers in the gap rather thanphosphorothioate DNA.

[0158] The language “non-activating region” includes a region of anantisense sequence, e.g., a chimeric oligonucleotide, that does notrecruit or activate RNase H. Preferably, a non-activating region doesnot comprise phosphorothioate DNA. The oligonucleotides of the inventioncomprise at least one non-activating region. In one embodiment, thenon-activating region can be stabilized against nucleases or can providespecificity for the target by being complementary to the target andforming hydrogen bonds with the target nucleic acid molecule, which isto be bound by the oligonucleotide.

[0159] Antisense sequences of the present invention may include“morpholino oligonucleotides.” Morpholino oligonucleotides are non-ionicand function by an RNase H-independent mechanism. Each of the 4 geneticbases (Adenine, Cytosine, Guanine, and Thymine/Uracil) of the morpholinooligonucleotides is linked to a 6-membered morpholine ring. Morpholinooligonucleotides are made by joining the 4 different subunit types by,e.g., non-ionic phosphorodiamidate inter-subunit linkages. An example ofa 2 subunit morpholino oligonucleotide is shown below.

[0160] Morpholino oligonucleotides have many advantages including:complete resistance to nucleases (Antisense & Nuc. Acid Drug Dev. 1996.6:267); predictable targeting (Biochemica Biophysica Acta. 1999.1489:141); reliable activity in cells (Antisense & Nuc. Acid Drug Dev.1997. 7:63); excellent sequence specificity (Antisense & Nuc. Acid DrugDev. 1997. 7:151); minimal non-antisense activity (Biochemica BiophysicaActa. 1999. 1489:141); and simple osmotic or scrape delivery (Antisense& Nuc. Acid Drug Dev. 1997. 7:291). Morpholino oligonucleotides are alsopreferred because of their non-toxicity at high doses. A discussion ofthe preparation of morpholino oligonucleotides can be found in Antisense& Nuc. Acid Drug Dev. 1997. 7:187.

[0161] Uptake Of Oligonucleotides By Cells

[0162] Oligonucleotides and oligonucleotide compositions are contactedwith (i.e., brought into contact with, also referred to herein asadministered or delivered to) and taken up by one or more cells or acell lysate. The term “cells” includes prokaryotic and eukaryotic cells,preferably vertebrate cells, and, more preferably, mammalian cells. In apreferred embodiment, the oligonucleotide compositions of the inventionare contacted with human cells.

[0163] Oligonucleotide compositions of the invention can be contactedwith cells in vitro, e.g., in a test tube or culture dish, (and may ormay not be introduced into a subject) or in vivo, e.g., in a subjectsuch as a mamalian subject. Oligonucleotides are taken up by cells at aslow rate by endocytosis, but endocytosed oligonucleotides are generallysequestered and not available, e.g., for hybridization to a targetnucleic acid molecule. In one embodiment, cellular uptake can befacilitated by electroporation or calcium phosphate precipitation.However, these procedures are only useful for in vitro or ex vivoembodiments, are not convenient and, in some cases, are associated withcell toxicity.

[0164] In another embodiment, delivery of oligonucleotides into cellscan be enhanced by suitable art recognized methods including calciumphosphate, DMSO, glycerol or dextran, electroporation, or bytransfection, e.g., using cationic, anionic, or neutral lipidcompositions or liposomes using methods known in the art (see e.g., WO90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No. 4,897,355; Bergan etal. 1993. Nucleic Acids Research. 21:3567). Enhanced delivery ofoligonucleotides can also be mediated by the use of vectors (See e.g.,Shi, Y. 2003. Trends Genet 2003 Jan 19:9; Reichhart J M et al. Genesis.2002. 34(1-2):160-4, Yu et al. 2002. Proc Natl Acad Sci U S A 99:6047;Sui et al. 2002. Proc Natl Acad Sci U S A 99:5515) viruses, polyamine orpolycation conjugates using compounds such as polylysine, protamine, orN1, N12-bis (ethyl) spermine (see, e.g., Bartzatt, R. et al. 1989.Biotechnol. Appl. Biochem. 11:133; Wagner E. et al. 1992. Proc. Natl.Acad. Sci. 88:4255)

[0165] Conjugating Agents

[0166] Conjugating agents bind to the oligonucleotide in a covalentmanner. In one embodiment, oligonucleotides can be derivitized orchemically modified by binding to a conjugating agent to facilitatecellular uptake. For example, covalent linkage of a cholesterol moietyto an oligonucleotide can improve cellular uptake by 5- to 10-fold whichin turn improves DNA binding by about 10-fold (Boutorin et al., 1989,FEBS Letters 254:129-132). Conjugation of octyl, dodecyl, and octadecylresidues enhances cellular uptake by 3-, 4-, and 10-fold as compared tounmodified oligonucleotides (Vlassov et al., 1994, Biochimica etBiophysica Acta 1197:95-108). Similarly, derivatization ofoligonucleotides with poly-L-lysine can aid oligonucleotide uptake bycells (Schell, 1974, Biochem. Biophys. Acta 340:323, and Lemaitre etal., 1987, Proc. Natl. Acad. Sci. USA 84:648).

[0167] Certain protein carriers can also facilitate cellular uptake ofoligonucleotides, including, for example, serum albumin, nuclearproteins possessing signals for transport to the nucleus, and viral orbacterial proteins capable of cell membrane penetration. Therefore,protein carriers are useful when associated with or linked to theoligonucleotides. Accordingly, the present invention provides forderivatization of oligonucleotides with groups capable of facilitatingcellular uptake, including hydrocarbons and non-polar groups,cholesterol, long chain alcohols (i.e., hexanol), poly-L-lysine andproteins, as well as other aryl or steroid groups and polycations havinganalogous beneficial effects, such as phenyl or naphthyl groups,quinoline, anthracene or phenanthracene groups, fatty acids, fattyalcohols and sesquiterpenes, diterpenes, and steroids. A major advantageof using conjugating agents is to increase the initial membraneinteraction that leads to a greater cellular accumulation ofoligonucleotides.

[0168] Encapsulating Agents

[0169] Encapsulating agents entrap oligonucleotides within vesicles. Inanother embodiment of the invention, an oligonucleotide may beassociated with a carrier or vehicle, e.g., liposomes or micelles,although other carriers could be used, as would be appreciated by oneskilled in the art. Liposomes are vesicles made of a lipid bilayerhaving a structure similar to biological membranes. Such carriers areused to facilitate the cellular uptake or targeting of theoligonucleotide, or improve the oligonucleotide's pharmacokinetic ortoxicologic properties.

[0170] For example, the oligonucleotides of the present invention mayalso be administered encapsulated in liposomes, pharmaceuticalcompositions wherein the active ingredient is contained either dispersedor variously present in corpuscles consisting of aqueous concentriclayers adherent to lipidic layers. The oligonucleotides, depending uponsolubility, may be present both in the aqueous layer and in the lipidiclayer, or in what is generally termed a liposomic suspension. Thehydrophobic layer, generally but not exclusively, comprises phopholipidssuch as lecithin and sphingomyelin, steroids such as cholesterol, moreor less ionic surfactants such as diacetylphosphate, stearylamine, orphosphatidic acid, or other materials of a hydrophobic nature. Thediameters of the liposomes generally range from about 15 nm to about 5microns.

[0171] The use of liposomes as drug delivery vehicles offers severaladvantages. Liposomes increase intracellular stability, increase uptakeefficiency and improve biological activity. Liposomes are hollowspherical vesicles composed of lipids arranged in a similar fashion asthose lipids which make up the cell membrane. They have an internalaqueous space for entrapping water soluble compounds and range in sizefrom 0.05 to several microns in diameter. Several studies have shownthat liposomes can deliver nucleic acids to cells and that the nucleicacids remain biologically active. For example, a liposome deliveryvehicle originally designed as a research tool, such as Lipofectin, candeliver intact nucleic acid molecules to cells.

[0172] Specific advantages of using liposomes include the following:they are non-toxic and biodegradable in composition; they display longcirculation half-lives; and recognition molecules can be readilyattached to their surface for targeting to tissues. Finally,cost-effective manufacture of liposome-based pharmaceuticals, either ina liquid suspension or lyophilized product, has demonstrated theviability of this technology as an acceptable drug delivery system.

[0173] Complexing Agents

[0174] Complexing agents bind to the oligonucleotides of the inventionby a strong but non-covalent attraction (e.g., an electrostatic, van derWaals, pi-stacking, etc. interaction). In one embodiment,oligonucleotides of the invention can be complexed with a complexingagent to increase cellular uptake of oligonucleotides. An example of acomplexing agent includes cationic lipids. Cationic lipids can be usedto deliver oligonucleotides to cells.

[0175] The term “cationic lipid” includes lipids and synthetic lipidshaving both polar and non-polar domains and which are capable of beingpositively charged at or around physiological pH and which bind topolyanions, such as nucleic acids, and facilitate the delivery ofnucleic acids into cells. In general cationic lipids include saturatedand unsaturated alkyl and alicyclic ethers and esters of amines, amides,or derivatives thereof. Straight-chain and branched alkyl and alkenylgroups of cationic lipids can contain, e.g., from 1 to about 25 carbonatoms. Preferred straight chain or branched alkyl or alkene groups havesix or more carbon atoms. Alicyclic groups include cholesterol and othersteroid groups. Cationic lipids can be prepared with a variety ofcounterions (anions) including, e.g., Cl⁻, Br⁻, I⁻, F⁻, acetate,trifluoroacetate, sulfate, nitrite, and nitrate.

[0176] Examples of cationic lipids include polyethylenimine,polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combinationof DOTMA and DOPE), Lipofectase, Lipofectamine, DOPE, Cytofectin (GileadSciences, Foster City, Calif.), and Eufectins (J B L, San Luis Obispo,Calif.). Exemplary cationic liposomes can be made fromN-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA),N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate(DOTAP), 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol(DC-Chol),2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; anddimethyldioctadecylammonium bromide (DDAB). The cationic lipidN-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),for example, was found to increase 1000-fold the antisense effect of aphosophorothioate oligonucleotide. (Vlassov et al., 1994, Biochimica etBiophysica Acta 1197:95-108). Oligonucleotides can also be complexedwith, e.g., poly (L-lysine) or avidin and lipids may, or may not, beincluded in this mixture, e.g., steryl-poly (L-lysine).

[0177] Cationic lipids have been used in the art to deliveroligonucleotides to cells (see, e.g., U.S. Pat. Nos. 5,855,910;5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al. 1996. Proc.Natl. Acad. Sci. USA 93:3176; Hope et al 1998. Molecular MembraneBiology 15:1). Other lipid compositions which can be used to facilitateuptake of the instant oligonucleotides can be used in connection withthe claimed methods. In addition to those listed supra, other lipidcompositions are also known in the art and include, e.g, those taught inU.S. Pat. No. 4,235,871, U.S. Pat. Nos. 4,501,728; 4,837,028; 4,737,323.

[0178] In one embodiment lipid compositions can further comprise agents,e.g., viral proteins to enhance lipid-mediated transfections ofoligonucleotides (Kamata, et al., 1994. Nucl. Acids. Res. 22:536). Inanother embodiment, oligonucleotides are contacted with cells as part ofa composition comprising an oligonucleotide, a peptide, and a lipid astaught, e.g., in U.S. Pat. No. 5,736,392. Improved lipids have also beendescribed which are serum resistant (Lewis, et al., 1996. Proc. Natl.Acad. Sci. 93:3176). Cationic lipids and other complexing agents act toincrease the number of oligonucleotides carried into the cell throughendocytosis.

[0179] In another embodiment N-substituted glycine oligonucleotides(peptoids) can be used to optimize uptake of oligonucleotides. Peptoidshave been used to create cationic lipid-like compounds for transfection(Murphy, et al., 1998. Proc. Natl. Acad. Sci. 95:1517). Peptoids can besynthesized using standard methods (e.g., Zuckermann, R. N., et al.1992. J. Am. Chem. Soc. 114:10646; Zuckermann, R. N., et al. 1992. Int.J Peptide Protein Res. 40:497). Combinations of cationic lipids andpeptoids, liptoids, can also be used to optimize uptake of the subjectoligonucleotides (Hunag, et al., 1998. Chemistry and Biology. 5:345).Liptoids can be synthesized by elaborating peptoid oligonucleotides andcoupling the amino terminal submonomer to a lipid via its amino group(Hunag, et al., 1998. Chemistry and Biology. 5:345).

[0180] It is known in the art that positively charged amino acids can beused for creating highly active cation lipids (Lewis et al. 1996. Proc.Natl. Acad. Sci. U.S.A. 93:3176). In one embodiment, a composition fordelivering oligonucleotides of the invention comprises a number ofarginine, lysine, histadine or ornithine residues linked to a lipophilicmoiety (see e.g., U.S. Pat. No. 5,777,153).

[0181] In another, a composition for delivering oligonucleotides of theinvention comprises a peptide having from between about one to aboutfour basic residues. These basic residues can be located, e.g., on theamino terminal, C-terminal, or internal region of the peptide. Familiesof amino acid residues having similar side chains have been defined inthe art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine (canalso be considered non-polar), asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Apart from the basic amino acids, a majority or all of theother residues of the peptide can be selected from the non-basic aminoacids, e.g., amino acids other than lysine, arginine, or histidine.Preferably a preponderance of neutral amino acids with long neutral sidechains are used. For example, a peptide such as (N-term)His-Ile-Trp-Leu-Ile-Tyr-Leu-Trp-Ile-Val-(C-term) (SEQ ID NO: ##) couldbe used. In one embodiment such a composition can be mixed with thefusogenic lipid DOPE as is well known in the art.

[0182] In one embodiment, the cells to be contacted with anoligonucleotide composition of the invention are contacted with amixture comprising the oligonucleotide and a mixture comprising a lipid,e.g., one of the lipids or lipid compositions described supra forbetween about 12 h to about 24 h. In another embodiment, the cells to becontacted with an oligonucleotide composition are contacted with amixture comprising the oligonucleotide and a mixture comprising a lipid,e.g., one of the lipids or lipid compositions described supra forbetween about 1 and about five days. In one embodiment, the cells arecontacted with a mixture comprising a lipid and the oligonucleotide forbetween about three days to as long as about 30 days. In anotherembodiment, a mixture comprising a lipid is left in contact with thecells for at least about five to about 20 days. In another embodiment, amixture comprising a lipid is left in contact with the cells for atleast about seven to about 15 days.

[0183] For example, in one embodiment, an oligonucleotide compositioncan be contacted with cells in the presence of a lipid such ascytofectin CS or GSV(available from Glen Research; Sterling, Va.),GS3815, GS2888 for prolonged incubation periods as described herein.

[0184] In one embodiment the incubation of the cells with the mixturecomprising a lipid and an oligonucleotide composition does not reducethe viability of the cells. Preferably, after the transfection periodthe cells are substantially viable. In one embodiment, aftertransfection, the cells are between at least about 70 and at least about100 percent viable. In another embodiment, the cells are between atleast about 80 and at least about 95% viable. In yet another embodiment,the cells are between at least about 85% and at least about 90% viable.

[0185] In one embodiment, oligonucleotides are modified by attaching apeptide sequence that transports the oligonucleotide into a cell,referred to herein as a “transporting peptide.” In one embodiment, thecomposition includes an oligonucleotide which is complementary to atarget nucleic acid molecule encoding the protein, and a covalentlyattached transporting peptide.

[0186] The language “transporting peptide” includes an amino acidsequence that facilitates the transport of an oligonucleotide into acell. Exemplary peptides which facilitate the transport of the moietiesto which they are linked into cells are known in the art, and include,e.g., HIV TAT transcription factor, lactoferrin, Herpes VP22 protein,and fibroblast growth factor 2 (Pooga et al. 1998. Nature Biotechnology.16:857; and Derossi et al. 1998. Trends in Cell Biology. 8:84; Elliottand O'Hare. 1997. Cell 88:223).

[0187] For example, in one embodiment, the transporting peptidecomprises an amino acid sequence derived from the antennapedia protein.Preferably, the peptide comprises amino acids 43-58 of the antennapediaprotein(Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys) (SEQID NO: ##) or a portion or variant thereof that facilitates transport ofan oligonucleotide into a cell (see, e.g., WO 91/1898; Derossi et al.1998. Trends Cell Biol. 8:84). Exemplary variants are shown in Derossiet al., supra.

[0188] In one embodiment, the transporting peptide comprises an aminoacid sequence derived from the transportan, galanin(1-12)-Lys-mastoparan (1-14) amide, protein. (Pooga et al. 1998. NatureBiotechnology 16:857). Preferably, the peptide comprises the amino acidsof the transportan protein shown in the sequenceGWTLNSAGYLLGKFNLKALAALAKKIL (SEQ ID NO: ##) or a portion or variantthereof that facilitates transport of an oligonucleotide into a cell.

[0189] In one embodiment, the transporting peptide comprises an aminoacid sequence derived from the HIV TAT protein. Preferably, the peptidecomprises amino acids 37-72 of the HIV TAT protein, e.g., shown in thesequence C(Acm)FITKALGISYGRKKRRQRRRPPQC (SEQ ID NO: ##) (TAT 37-60;where C(Acm) is Cys-acetamidomethyl) or a portion or variant thereof,e.g., C(Acm)GRKKRRQRRRPPQC (SEQ ID NO: ##)(TAT 48-40) orC(Acm)LGISYGRKKRRQRRPPQC (SEQ ID NO: ##) (TAT 43-60) that facilitatestransport of an oligonucleotide into a cell (Vives et al. 1997. J. Biol.Chem. 272:16010). In another embodiment the peptide(G)CFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ (SEQ ID NO: ##) can be used.

[0190] Portions or variants of transporting peptides can be readilytested to determine whether they are equivalent to these peptideportions by comparing their activity to the activity of the nativepeptide, e.g., their ability to transport fluorescently-labeledoligonucleotides to cells. Fragments or variants that retain the abilityof the native transporting peptide to transport an oligonucleotide intoa cell are functionally equivalent and can be substituted for the nativepeptides.

[0191] Oligonucleotides can be attached to the transporting peptideusing known techniques, e.g., ( Prochiantz, A. 1996. Curr. Opin.Neurobiol. 6:629; Derossi et al. 1998. Trends Cell Biol. 8:84; Troy etal. 1996. J. Neurosci. 16:253), Vives et al. 1997. J. Biol. Chem.272:16010). For example, in one embodiment, oligonucleotides bearing anactivated thiol group are linked via that thiol group to a cysteinepresent in a transport peptide (e.g., to the cysteine present in the βturn between the second and the third helix of the antennapediahomeodomain as taught, e.g., in Derossi et al. 1998. Trends Cell Biol.8:84; Prochiantz. 1996. Current Opinion in Neurobiol. 6:629; Allinquantet al. 1995. J Cell Biol. 128:919). In another embodiment, aBoc-Cys-(Npys)OH group can be coupled to the transport peptide as thelast (N-terminal) amino acid and an oligonucleotide bearing an SH groupcan be coupled to the peptide (Troy et al. 1996. J. Neurosci. 16:253).

[0192] In one embodiment, a linking group can be attached to anucleomonomer and the transporting peptide can be covalently attached tothe linker. In one embodiment, a linker can function as both anattachment site for a transporting peptide and can provide stabilityagainst nucleases. Examples of suitable linkers include substituted orunsubstituted C₁-C₂₀ alkyl chains, C₂-C₂₀ alkenyl chains, C₂-C₂₀ alkynylchains, peptides, and heteroatoms (e.g., S, O, NH, etc.). Otherexemplary linkers include bifunctional crosslinking agents such assulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g., Smithet al. Biochem J 1991. 276: 417-2).

[0193] In one embodiment, oligonucleotides of the invention aresynthesized as molecular conjugates which utilize receptor-mediatedendocytotic mechanisms for delivering genes into cells (see, e.g.,Bunnell et al. 1992. Somatic Cell and Molecular Genetics. 18:559 and thereferences cited therein).

[0194] Targeting Agents

[0195] The delivery of oligonucleotides can also be improved bytargeting the oligonucleotides to a cellular receptor. The targetingmoieties can be conjugated to the oligonucleotides or attached to acarrier group (i.e., poly(L-lysine) or liposomes) linked to theoligonucleotides. This method is well suited to cells that displayspecific receptor-mediated endocytosis.

[0196] For instance, oligonucleotide conjugates to 6-phosphomannosylatedproteins are internalized 20-fold more efficiently by cells expressingmannose 6-phosphate specific receptors than free oligonucleotides. Theoligonucleotides may also be coupled to a ligand for a cellular receptorusing a biodegradable linker. In another example, the delivery constructis mannosylated streptavidin which forms a tight complex withbiotinylated oligonucleotides. Mannosylated streptavidin was found toincrease 20-fold the internalization of biotinylated oligonucleotides.(Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).

[0197] In addition specific ligands can be conjugated to the polylysinecomponent of polylysine-based delivery systems. For example,transferrin-polylysine, adenovirus-polylysine, and influenza virushemagglutinin HA-2 N-terminal fusogenic peptides-polylysine conjugatesgreatly enhance receptor-mediated DNA delivery in eucaryotic cells.Mannosylated glycoprotein conjugated to poly(L-lysine) in aveolarmacrophages has been employed to enhance the cellular uptake ofoligonucleotides. Liang et al. 1999. Pharmazie 54:559-566.

[0198] Because malignant cells have an increased need for essentialnutrients such as folic acid and transferrin, these nutrients can beused to target oligonucleotides to cancerous cells. For example, whenfolic acid is linked to poly(L-lysine) enhanced oligonucleotide uptakeis seen in promyelocytic leukaemia (HL-60) cells and human melanoma(M-14) cells. Ginobbi et al. 1997. Anticancer Res. 17:29. In anotherexample, liposomes coated with maleylated bovine serum albumin, folicacid, or ferric protoporphyrin IX, show enhanced cellular uptake ofoligonucleotides in murine macrophages, KB cells, and 2.2.15 humanhepatoma cells. Liang et al. 1999. Pharmazie 54:559-566.

[0199] Liposomes naturally accumulate in the liver, spleen, andreticuloendothelial system (so-called, passive targeting). By couplingliposomes to various ligands such as antibodies are protein A, they canbe actively targeted to specific cell populations. For example, proteinA-bearing liposomes may be pretreated with H-2K specific antibodieswhich are targeted to the mouse major histocompatibility complex-encodedH-2K protein expressed on L cells. (Vlassov et al. 1994. Biochimica etBiophysica Acta 1197:95-108).

[0200] Assays of Oligonucleotide Stability

[0201] Preferably, the double-stranded oligonucleotides of the inventionare stabilized, i.e., substantially resistant to endonuclease andexonuclease degradation. An oligonucleotide is defined as beingsubstantially resistant to nucleases when it is at least about 3-foldmore resistant to attack by an endogenous cellular nuclease, and ishighly nuclease resistant when it is at least about 6-fold moreresistant than a corresponding, single-stranded oligonucleotide. Thiscan be demonstrated by showing that the oligonucleotides of theinvention are substantially resist nucleases using techniques which areknown in the art.

[0202] One way in which substantial stability can be demonstrated is byshowing that the oligonucleotides of the invention function whendelivered to a cell, e.g., that they reduce transcription or translationof target nucleic acid molecules, e.g., by measuring protein levels orby measuring cleavage of mRNA. Assays which measure the stability oftarget RNA can be performed at about 24 hours post-transfection (e.g.,using Northern blot techniques, RNase Protection Assays, or QC-PCRassays as known in the art). Alternatively, levels of the target proteincan be measured. Preferably, in addition to testing the RNA or proteinlevels of interest, the RNA or protein levels of a control, non-targetedgene will be measured (e.g., actin, or preferably a control withsequence similarity to the target) as a specificity control. RNA orprotein measurements can be made using any art-recognized technique.Preferably, measurements will be made beginning at about 16-24 hourspost transfection. (M. Y. Chiang, et al. 1991. J. Biol Chem.266:18162-71; T. Fisher, et al. 1993. Nucleic Acids Research. 21 3857).

[0203] The ability of an oligonucleotide composition of the invention toinhibit protein synthesis can be measured using techniques which areknown in the art, for example, by detecting an inhibition in genetranscription or protein synthesis. For example, Nuclease S1 mapping canbe performed. In another example, Northern blot analysis can be used tomeasure the presence of RNA encoding a particular protein. For example,total RNA can be prepared over a cesium chloride cushion (see, e.g.,Ausebel et al., 1987. Current Protocols in Molecular Biology (Greene &Wiley, New York)). Northern blots can then be made using the RNA andprobed (see, e.g., Id.). In another example, the level of the specificmRNA produced by the target protein can be measured, e.g., using PCR. Inyet another example, Western blots can be used to measure the amount oftarget protein present. In still another embodiment, a phenotypeinfluenced by the amount of the protein can be detected. Techniques forperforming Western blots are well known in the art, see, e.g., Chen etal. J. Biol. Chem. 271:28259.

[0204] In another example, the promoter sequence of a target gene can belinked to a reporter gene and reporter gene transcription (e.g., asdescribed in more detail below) can be monitored. Alternatively,oligonucleotide compositions that do not target a promoter can beidentified by fusing a portion of the target nucleic acid molecule witha reporter gene so that the reporter gene is transcribed. By monitoringa change in the expression of the reporter gene in the presence of theoligonucleotide composition, it is possible to determine theeffectiveness of the oligonucleotide composition in inhibiting theexpression of the reporter gene. For example, in one embodiment, aneffective oligonucleotide composition will reduce the expression of thereporter gene.

[0205] A “reporter gene” is a nucleic acid that expresses a detectablegene product, which may be RNA or protein. Detection of mRNA expressionmay be accomplished by Northern blotting and detection of protein may beaccomplished by staining with antibodies specific to the protein.Preferred reporter genes produce a readily detectable product. Areporter gene may be operably linked with a regulatory DNA sequence suchthat detection of the reporter gene product provides a measure of thetranscriptional activity of the regulatory sequence. In preferredembodiments, the gene product of the reporter gene is detected by anintrinsic activity associated with that product. For instance, thereporter gene may encode a gene product that, by enzymatic activity,gives rise to a detectable signal based on color, fluorescence, orluminescence. Examples of reporter genes include, but are not limitedto, those coding for chloramphenicol acetyl transferase (CAT),luciferase, β-galactosidase, and alkaline phosphatase.

[0206] One skilled in the art would readily recognize numerous reportergenes suitable for use in the present invention. These include, but arenot limited to, chloramphenicol acetyltransferase (CAT), luciferase,human growth hormone (hGH), and beta-galactosidase. Examples of suchreporter genes can be found in F. A. Ausubel et al., Eds., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, (1989). Anygene that encodes a detectable product, e.g., any product havingdetectable enzymatic activity or against which a specific antibody canbe raised, can be used as a reporter gene in the present methods.

[0207] One reporter gene system is the firefly luciferase reportersystem. (Gould, S. J., and Subramani, S. 1988. Anal. Biochem., 7:404-408incorporated herein by reference). The luciferase assay is fast andsensitive. In this assay, a lysate of the test cell is prepared andcombined with ATP and the substrate luciferin. The encoded enzymeluciferase catalyzes a rapid, ATP dependent oxidation of the substrateto generate a light-emitting product. The total light output is measuredand is proportional to the amount of luciferase present over a widerange of enzyme concentrations.

[0208] CAT is another frequently used reporter gene system; a majoradvantage of this system is that it has been an extensively validatedand is widely accepted as a measure of promoter activity. (Gorman C. M.,Moffat, L. F., and Howard, B. H. 1982. Mol. Cell. Biol., 2:1044-1051).In this system, test cells are transfected with CAT expression vectorsand incubated with the candidate substance within 2-3 days of theinitial transfection. Thereafter, cell extracts are prepared. Theextracts are incubated with acetyl CoA and radioactive chloramphenicol.Following the incubation, acetylated chloramphenicol is separated fromnonacetylated form by thin layer chromatography. In this assay, thedegree of acetylation reflects the CAT gene activity with the particularpromoter.

[0209] Another suitable reporter gene system is based on immunologicdetection of hGH. This system is also quick and easy to use. (Selden,R., Burke-Howie, K. Rowe, M. E., Goodman, H. M., and Moore, D. D.(1986), Mol. Cell, Biol., 6:3173-3179 incorporated herein by reference).The hGH system is advantageous in that the expressed hGH polypeptide isassayed in the media, rather than in a cell extract. Thus, this systemdoes not require the destruction of the test cells. It will beappreciated that the principle of this reporter gene system is notlimited to hGH but rather adapted for use with any polypeptide for whichan antibody of acceptable specificity is available or can be prepared.

[0210] In one embodiment, nuclease stability of a double-strandedoligonucleotide of the invention is measured and compared to a control,e.g., an RNAi molecule typically used in the art (e.g., a duplexoligonucleotide of less than 25 nucleotides in length and comprising 2nucleotide base overhangs) or an unmodified RNA duplex with blunt ends.

[0211] Oligonucleotide Synthesis

[0212] Oligonucleotides of the invention can be synthesized by anymethod known in the art, e.g., using enzymatic synthesis and chemicalsynthesis. The oligonucleotides can be synthesized in vitro (e.g., usingenzymatic synthesis and chemical synthesis) or in vivo (usingrecombinant DNA technology well known in the art).

[0213] In a preferred embodiment, chemical synthesis is used. Chemicalsynthesis of linear oligonucleotides is well known in the art and can beachieved by solution or solid phase techniques. Preferably, synthesis isby solid phase methods. Oligonucleotides can be made by any of severaldifferent synthetic procedures including the phosphoramidite, phosphitetriester, H-phosphonate, and phosphotriester methods, typically byautomated synthesis methods.

[0214] Oligonucleotide synthesis protocols are well known in the art andcan be found, e.g., in U.S. Pat. No. 5,830,653; WO 98/13526; Stec et al.1984. J. Am. Chem. Soc. 106:6077; Stec et al. 1985. J. Org. Chem.50:3908; Stec et al. J. Chromatog. 1985. 326:263; LaPlanche et al. 1986.Nuc. Acid. Res. 1986. 14:9081; Fasman G. D., 1989. Practical Handbook ofBiochemistry and Molecular Biology. 1989. CRC Press, Boca Raton, Fla.;Lamone. 1993. Biochem. Soc. Trans. 21:1; U.S. Pat. No. 5,013,830; U.S.Pat. No. 5,214,135; U.S. Pat. No. 5,525,719; Kawasaki et al. 1993. J.Med. Chem. 36:831; WO 92/03568; U.S. Pat. No. 5,276,019; U.S. Pat. No.5,264,423.

[0215] The synthesis method selected can depend on the length of thedesired oligonucleotide and such choice is within the skill of theordinary artisan. For example, the phosphoramidite and phosphitetriester method can produce oligonucleotides having 175 or morenucleotides while the H-phosphonate method works well foroligonucleotides of less than 100 nucleotides. If modified bases areincorporated into the oligonucleotide, and particularly if modifiedphosphodiester linkages are used, then the synthetic procedures arealtered as needed according to known procedures. In this regard, Uhlmannet al. (1990, Chemical Reviews 90:543-584) provide references andoutline procedures for making oligonucleotides with modified bases andmodified phosphodiester linkages. Other exemplary methods for makingoligonucleotides are taught in Sonveaux. 1994. “Protecting Groups inOligonucleotide Synthesis”; Agrawal. Methods in Molecular Biology 26:1.Exemplary synthesis methods are also taught in “OligonucleotideSynthesis—A Practical Approach” (Gait, M. J. IRL Press at OxfordUniversity Press. 1984). Moreover, linear oligonucleotides of definedsequence, including some sequences with modified nucleotides, arereadily available from several commercial sources.

[0216] The oligonucleotides may be purified by polyacrylamide gelelectrophoresis, or by any of a number of chromatographic methods,including gel chromatography and high pressure liquid chromatography. Toconfirm a nucleotide sequence, oligonucleotides may be subjected to DNAsequencing by any of the known procedures, including Maxam and Gilbertsequencing, Sanger sequencing, capillary electrophoresis sequencing thewandering spot sequencing procedure or by using selective chemicaldegradation of oligonucleotides bound to Hybond paper. Sequences ofshort oligonucleotides can also be analyzed by laser desorption massspectroscopy or by fast atom bombardment (McNeal, et al., 1982, J. Am.Chem. Soc. 104:976; Viari, et al., 1987, Biomed. Environ. Mass Spectrom.14:83; Grotjahn et al., 1982, Nuc. Acid Res. 10:4671). Sequencingmethods are also available for RNA oligonucleotides.

[0217] The quality of oligonucleotides synthesized can be verified bytesting the oligonucleotide by capillary electrophoresis and denaturingstrong anion HPLC (SAX-HPLC) using, e.g., the method of Bergot and Egan.1992. J. Chrom. 599:35.

[0218] Other exemplary synthesis techniques are well known in the art(see, e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual,Second Edition (1989); DNA Cloning, Volumes I and II (D N Glover Ed.1985); Oligonucleotide Synthesis (M J Gait Ed, 1984; Nucleic AcidHybridisation (B D Hames and S J Higgins eds. 1984); A Practical Guideto Molecular Cloning (1984); or the series, Methods in Enzymology(Academic Press, Inc.)).

[0219] Uses of Oligonucleotides

[0220] This invention also features methods of inhibiting expression ofa protein in a cell including contacting the cell with one of theabove-described oligonucleotide compositions.

[0221] The oligonucleotides of the invention can be used in a variety ofin vitro and in vivo situations to specifically inhibit proteinexpression. The instant methods and compositions are suitable for bothin vitro and in vivo use.

[0222] The methods of the invention may be used for determining thefunction of a gene in a cell or an organism or for modulating thefunction of a gene in a cell or an organism, being capable of respondingto or mediating RNA interference. The cell is preferably a eukaryoticcell or a cell line, e.g., an animal cell such as a mammalian cell,e.g., an embryonic cell, a pluripotent stem cell, a tumor cell, e.g., ateratocarcinoma cell, or a virus-infected cell. The organism ispreferably a eukaryotic organism, e.g., an animal such as a mammal,particularly a human.

[0223] The invention includes methods to inhibit expression of a targetgene in a cell in vitro. For example, such methods may includeintroduction of RNA into a cell in an amount sufficient to inhibitexpression of the target gene, where the RNA is a double-strandedmolecule of the invention. By way of a further example, such an RNAmolecule may have a first strand consisting essentially of aribonucleotide sequence that corresponds to a nucleotide sequence of thetarget gene, and a second strand consisting essentially of aribonucleotide sequence that is complementary to the nucleotide sequenceof the target gene, in which the first and the second strands areseparate complementary strands or are joined by a loop, and theyhybridize to each other to form said double-stranded molecule, such thatthe duplex composition inhibits expression of the target gene. Theduplex composition may include modified nucleomonomers as discussedabove.

[0224] The invention also relates to a method to inhibit expression of atarget gene in an invertebrate organism. Such methods include providingan invertebrate organism containing a target cell that contains thetarget gene, in which the target cell is susceptible to RNA interferenceand the target gene is expressed in the target cell. Such methodsfurther include contacting the invertebrate organism with an RNAcomposition of the invention. For example, the RNA may be adouble-stranded molecule with a first strand consisting essentially of aribonucleotide sequence that corresponds to a nucleotide sequence of thetarget gene and a second strand consisting essentially of aribonucleotide sequence that is complementary to the nucleotide sequenceof the target gene. In such cases, the first and the secondribonucleotide sequences may be separate complementary strands or joinedby a loop, and they hybridize to each other to form the double-strandedmolecule. Finally, such methods include a step of introducing the duplexRNA composition into the target cell to thereby inhibiting expression ofthe target gene.

[0225] In one embodiment, the oligonucleotides of the invention can beused to inhibit gene function in vitro in a method for identifying thefunctions of genes. In this manner, the transcription of genes that areidentified, but for which no function has yet been shown, can beinhibited to thereby determine how the phenotype of a cell is changedwhen the gene is not transcribed. Such methods are useful for thevalidation of genes as targets for clinical treatment, e.g., witholigonucleotides or with other therapies.

[0226] To determine the effect of a composition of the invention, avariety of end points can be used. In addition to the assays describedpreviously herein, for example, nucleic acid probes (e.g., in the formof arrays) can be used to evaluate transcription patterns produced bycells. Probes can also be used detect peptides, proteins, or proteindomains, e.g., antibodies can be used to detect the expression of aparticular protein. In yet another embodiment, the function of a protein(e.g., enzymatic activity) can be measured. In yet another embodiment,the phenotype of a cell can be evaluated to determine whether or not atarget protein is expressed. For example, the ability of a compositionto affect a phenotype of a cell that is associated with cancer can betested.

[0227] In one embodiment, one or more additional agents (e.g.,activating agents, inducing agents, proliferation enhancing agents,tumor promoters) can be added to the cells.

[0228] In another embodiment, the compositions of the invention can beused to monitor biochemical reactions such as, e.g., interactions ofproteins, nucleic acids, small molecules, or the like, for example theefficiency or specificity of interactions between antigens andantibodies; or of receptors (such as purified receptors or receptorsbound to cell membranes) and their ligands, agonists or antagonists; orof enzymes (such as proteases or kinases) and their substrates, orincreases or decreases in the amount of substrate converted to aproduct; as well as many others. Such biochemical assays can be used tocharacterize properties of the probe or target, or as the basis of ascreening assay. For example, to screen samples for the presence ofparticular proteases (e.g., proteases involved in blood clotting such asproteases Xa and VIIa), the samples can be assayed, for example usingprobes which are fluorogenic substrates specific for each protease ofinterest. If a target protease binds to and cleaves a substrate, thesubstrate will fluoresce, usually as a result, e.g., of cleavage andseparation between two energy transfer pairs, and the signal can bedetected. In another example, to screen samples for the presence of aparticular kinase(s) (e.g., a tyrosine kinase), samples containing oneor more kinases of interest can be assayed, e.g., using probes arepeptides which can be selectively phosphorylated by one of the kinasesof interest. Using art-recognized, routinely determinable conditions,samples can be incubated with an array of substrates, in an appropriatebuffer and with the necessary cofactors, for an empirically determinedperiod of time. If necessary, reactions can be stopped, e.g., by washingand the phosphorylated substrates can be detected by, for example,incubating them with detectable reagents such as, e.g.,fluorescein-labeled anti-phosphotyrosine or anti-phosphoserineantibodies and the signal can be detected.

[0229] In another embodiment, the compositions of the invention can beused to screen for agents which modulate a pattern of gene expression.Arrays of oligonucleotides can be used, for example, to identify mRNAspecies whose pattern of expression from a set of genes is correlatedwith a particular physiological state or developmental stage, or with adisease condition (“correlative” genes, RNAs, or expression patterns).By the terms “correlate” or “correlative,” it is meant that thesynthesis pattern of RNA is associated with the physiological conditionof a cell, but not necessarily that the expression of a given RNA isresponsible for or is causative of a particular physiological state. Forexample, a small subset of mRNAs can be identified which are modulated(e.g., upregulated or downregulated) in cells which serve as a model fora particular disease state. This altered pattern of expression ascompared to that in a normal cell, which does not exhibit a pathologicalphenotype, can serve as a indicator of the disease state (“indicator” or“correlatvie” genes, RNAs, or expression patterns).

[0230] Compositions which modulate the chosen indicator expressionpattern (e.g., compared to control compositions comprising, for exampleoligonucleotides which comprise a nucleotide sequence which is thereverse of the oligonucleotide, or which contains mismatch bases) canindicate that a particular target gene is a potential target fortherapeutic intervention. Moreover, such compositions may be useful astherapeutic agents to modulate expression patters of cells in an invitro expression system or in in vivo therapy. As used herein,“modulate” means to cause to increase or decrease the amount or activityof a molecule or the like which is involved in a measurable reaction. Inone embodiment, a series of cells (e.g., from a disease model) can becontacted with a series of agents (e.g., for a period of time rangingfrom about 10 minutes to about 48 hours or more) and, using routine,art-recognized methods (e.g., commercially available kits), total RNA ormRNA extracts can be made. If it is desired to amplify the amount ofRNA, standard procedures such as RT-PCR amplification can be used (see,e.g., Innis et al eds., (1996) PCR Protocols: A Guide to Methods inAmplification, Academic Press, New York). The extracts (or amplifiedproducts from them) can be allowed to contact (e.g., incubate with)probes for appropriate indicator RNAs, and those agents which areassociated with a change in the indicator expression pattern can beidentified.

[0231] Similarly, agents can be identified which modulate expressionpatterns associated with particular physiological states ordevelopmental stages. Such agents can be man-made or naturally-occurringsubstances, including environmental factors such as substances involvedin embryonic development or in regulating physiological reactions.

[0232] In one embodiment, the methods described herein can be performedin a “high throughput” manner, in which a large number of target genes(e.g., as many as about 1000 or more, depending on the particular formatused) are assayed rapidly and concurrently. Further, many assay formats(e.g., plates or surfaces) can be processed at one time. For example,because the oligonucleotides of the invention do not need to be testedindividually before incorporating them into a composition, they can bereadily synthesized and large numbers of target genes can be tested atone time. For example, a large number of samples, each comprising abiological sample containing a target nucleic acid molecule (e.g., acell) and a composition of the invention can be added to separateregions of an assay format and assays can be performed on each of thesamples.

[0233] Administration of Oligonucleotide Compositions

[0234] The optimal course of administration or delivery of theoligonucleotides may vary depending upon the desired result and/or onthe subject to be treated. As used herein “administration” refers tocontacting cells with oligonucleotides and can be performed in vitro orin vivo. The dosage of oligonucleotides may be adjusted to optimallyreduce expression of a protein translated from a target nucleic acidmolecule, e.g., as measured by a readout of RNA stability or by atherapeutic response, without undue experimentation.

[0235] For example, expression of the protein encoded by the nucleicacid target can be measured to determine whether or not the dosageregimen needs to be adjusted accordingly. In addition, an increase ordecrease in RNA or protein levels in a cell or produced by a cell can bemeasured using any art recognized technique. By determining whethertranscription has been decreased, the effectiveness of theoligonucleotide in inducing the cleavage of a target RNA can bedetermined.

[0236] Any of the above-described oligonucleotide compositions can beused alone or in conjunction with a pharmaceutically acceptable carrier.As used herein, “pharmaceutically acceptable carrier” includesappropriate solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, it can beused in the therapeutic compositions. Supplementary active ingredientscan also be incorporated into the compositions.

[0237] Oligonucleotides may be incorporated into liposomes or liposomesmodified with polyethylene glycol or admixed with cationic lipids forparenteral administration. Incorporation of additional substances intothe liposome, for example, antibodies reactive against membrane proteinsfound on specific target cells, can help target the oligonucleotides tospecific cell types.

[0238] Moreover, the present invention provides for administering thesubject oligonucleotides with an osmotic pump providing continuousinfusion of such oligonucleotides, for example, as described inRataiczak et al. (1992 Proc. Natl. Acad. Sci. USA 89:11823-11827). Suchosmotic pumps are commercially available, e.g., from Alzet Inc. (PaloAlto, Calif.). Topical administration and parenteral administration in acationic lipid carrier are preferred.

[0239] With respect to in vivo applications, the formulations of thepresent invention can be administered to a patient in a variety of formsadapted to the chosen route of administration, e.g., parenterally,orally, or intraperitoneally. Parenteral administration, which ispreferred, includes administration by the following routes: intravenous;intramuscular; interstitially; intraarterially; subcutaneous; intraocular; intrasynovial; trans epithelial, including transdermal;pulmonary via inhalation; ophthalmic; sublingual and buccal; topically,including ophthalmic; dermal; ocular; rectal; and nasal inhalation viainsufflation.

[0240] Pharmaceutical preparations for parenteral administration includeaqueous solutions of the active compounds in water-soluble orwater-dispersible form. In addition, suspensions of the active compoundsas appropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol, or dextran, optionally, thesuspension may also contain stabilizers. The oligonucleotides of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the oligonucleotides may be formulated in solidform and redissolved or suspended immediately prior to use. Lyophilizedforms are also included in the invention.

[0241] Pharmaceutical preparations for topical administration includetransdermal patches, ointments, lotions, creams, gels, drops, sprays,suppositories, liquids and powders. In addition, conventionalpharmaceutical carriers, aqueous, powder or oily bases, or thickenersmay be used in pharmaceutical preparations for topical administration.

[0242] Pharmaceutical preparations for oral administration includepowders or granules, suspensions or solutions in water or non-aqueousmedia, capsules, sachets or tablets. In addition, thickeners, flavoringagents, diluents, emulsifiers, dispersing aids, or binders may be usedin pharmaceutical preparations for oral administration.

[0243] For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are known in the art, and include, for example, fortransmucosal administration bile salts and fusidic acid derivatives, anddetergents. Transmucosal administration may be through nasal sprays orusing suppositories. For oral administration, the oligonucleotides areformulated into conventional oral administration forms such as capsules,tablets, and tonics. For topical administration, the oligonucleotides ofthe invention are formulated into ointments, salves, gels, or creams asknown in the art.

[0244] Drug delivery vehicles can be chosen e.g., for in vitro, forsystemic, or for topical administration. These vehicles can be designedto serve as a slow release reservoir or to deliver their contentsdirectly to the target cell. An advantage of using some direct deliverydrug vehicles is that multiple molecules are delivered per uptake. Suchvehicles have been shown to increase the circulation half-life of drugsthat would otherwise be rapidly cleared from the blood stream. Someexamples of such specialized drug delivery vehicles which fall into thiscategory are liposomes, hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesive microspheres.

[0245] The described oligonucleotides may be administered systemicallyto a subject. Systemic absorption refers to the entry of drugs into theblood stream followed by distribution throughout the entire body.Administration routes which lead to systemic absorption include:intravenous, subcutaneous, intraperitoneal, and intranasal. Each ofthese administration routes delivers the oligonucleotide to accessiblediseased cells. Following subcutaneous administration, the therapeuticagent drains into local lymph nodes and proceeds through the lymphaticnetwork into the circulation. The rate of entry into the circulation hasbeen shown to be a function of molecular weight or size. The use of aliposome or other drug carrier localizes the oligonucleotide at thelymph node. The oligonucleotide can be modified to diffuse into thecell, or the liposome can directly participate in the delivery of eitherthe unmodified or modified oligonucleotide into the cell.

[0246] The chosen method of delivery will result in entry into cells.Preferred delivery methods include liposomes (10-400 nm), hydrogels,controlled-release polymers, and other pharmaceutically applicablevehicles, and microinjection or electroporation (for ex vivotreatments).

[0247] The pharmaceutical preparations of the present invention may beprepared and formulated as emulsions. Emulsions are usually heterogenoussystems of one liquid dispersed in another in the form of dropletsusually exceeding 0.1 μm in diameter.

[0248] The emulsions of the present invention may contain excipientssuch as emulsifiers, stabilizers, dyes, fats, oils, waxes, fatty acids,fatty alcohols, fatty esters, humectants, hydrophilic colloids,preservatives, and anti-oxidants may also be present in emulsions asneeded. These excipients may be present as a solution in either theaqueous phase, oily phase or itself as a separate phase.

[0249] Examples of naturally occurring emulsifiers that may be used inemulsion formulations of the present invention include lanolin, beeswax,phosphatides, lecithin and acacia. Finely divided solids have also beenused as good emulsifiers especially in combination with surfactants andin viscous preparations. Examples of finely divided solids that may beused as emulsifiers include polar inorganic solids, such as heavy metalhydroxides, nonswelling clays such as bentonite, attapulgite, hectorite,kaolin, montmorillonite, colloidal aluminum silicate and colloidalmagnesium aluminum silicate, pigments and nonpolar solids such as carbonor glyceryl tristearate.

[0250] Examples of preservatives that may be included in the emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Examples of antioxidants that may be included in the emulsionformulations include free radical scavengers such as tocopherols, alkylgallates, butylated hydroxyanisole, butylated hydroxytoluene, orreducing agents such as ascorbic acid and sodium metabisulfite, andantioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0251] In one embodiment, the compositions of oligonucleotides areformulated as microemulsions. A microemulsion is a system of water, oiland amphiphile which is a single optically isotropic andthermodynamically stable liquid solution. Typically microemulsions areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a 4th component, generally anintermediate chain-length alcohol to form a transparent system.

[0252] Surfactants that may be used in the preparation of microemulsionsinclude, but are not limited to, ionic surfactants, non-ionicsurfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fattyacid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate(MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate(PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate(MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate(DA0750), alone or in combination with cosurfactants. The cosurfactant,usually a short-chain alcohol such as ethanol, 1-propanol, and1-butanol, serves to increase the interfacial fluidity by penetratinginto the surfactant film and consequently creating a disordered filmbecause of the void space generated among surfactant molecules.

[0253] Microemulsions may, however, be prepared without the use ofcosurfactants and alcohol-free self-emulsifying microemulsion systemsare known in the art. The aqueous phase may typically be, but is notlimited to, water, an aqueous solution of the drug, glycerol, PEG300,PEG400, polyglycerols, propylene glycols, and derivatives of ethyleneglycol. The oil phase may include, but is not limited to, materials suchas Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain(C₈-C₁₂) mono, di, and tri-glycerides, polyoxyethylated glyceryl fattyacid esters, fatty alcohols, polyglycolized glycerides, saturatedpolyglycolized C₈-C₁₀ glycerides, vegetable oils and silicone oil.

[0254] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both oil/water and water/oil) have been proposed toenhance the oral bioavailability of drugs.

[0255] Microemulsions offer improved drug solubilization, protection ofdrug from enzymatic hydrolysis, possible enhancement of drug absorptiondue to surfactant-induced alterations in membrane fluidity andpermeability, ease of preparation, ease of oral administration oversolid dosage forms, improved clinical potency, and decreased toxicity(Constantinides et al., Pharmaceutical Research, 1994, 11:1385; Ho etal., J. Pharm. Sci., 1996, 85:138-143). Microemulsions have also beeneffective in the transdermal delivery of active components in bothcosmetic and pharmaceutical applications. It is expected that themicroemulsion compositions and formulations of the present inventionwill facilitate the increased systemic absorption of oligonucleotidesfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides within the gastrointestinal tract, vagina,buccal cavity and other areas of administration.

[0256] In an embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Evennon-lipophilic drugs may cross cell membranes if the membrane to becrossed is treated with a penetration enhancer. In addition toincreasing the diffusion of non-lipophilic drugs across cell membranes,penetration enhancers also act to enhance the permeability of lipophilicdrugs.

[0257] Five categories of penetration enhancers that may be used in thepresent invention include: surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants. Other agents may beutilized to enhance the penetration of the administered oligonucleotidesinclude: glycols such as ethylene glycol and propylene glycol, pyrrolssuch as 2-15 pyrrol, azones, and terpenes such as limonene, andmenthone.

[0258] The oligonucleotides, especially in lipid formulations, can alsobe administered by coating a medical device, for example, a catheter,such as an angioplasty balloon catheter, with a cationic lipidformulation. Coating may be achieved, for example, by dipping themedical device into a lipid formulation or a mixture of a lipidformulation and a suitable solvent, for example, an aqueous-basedbuffer, an aqueous solvent, ethanol, methylene chloride, chloroform andthe like. An amount of the formulation will naturally adhere to thesurface of the device which is subsequently administered to a patient,as appropriate. Alternatively, a lyophilized mixture of a lipidformulation may be specifically bound to the surface of the device. Suchbinding techniques are described, for example, in K. Ishihara et al.,Journal of Biomedical Materials Research, Vol. 27, pp. 1309-1314 (1993),the disclosures of which are incorporated herein by reference in theirentirety.

[0259] The useful dosage to be administered and the particular mode ofadministration will vary depending upon such factors as the cell type,or for in vivo use, the age, weight and the particular animal and regionthereof to be treated, the particular oligonucleotide and deliverymethod used, the therapeutic or diagnostic use contemplated, and theform of the formulation, for example, suspension, emulsion, micelle orliposome, as will be readily apparent to those skilled in the art.Typically, dosage is administered at lower levels and increased untilthe desired effect is achieved. When lipids are used to deliver theoligonucleotides, the amount of lipid compound that is administered canvary and generally depends upon the amount of oligonucleotide agentbeing administered. For example, the weight ratio of lipid compound tooligonucleotide agent is preferably from about 1:1 to about 15:1, with aweight ratio of about 5:1 to about 10:1 being more preferred. Generally,the amount of cationic lipid compound which is administered will varyfrom between about 0.1 milligram (mg) to about 1 gram (g). By way ofgeneral guidance, typically between about 0.1 mg and about 10 mg of theparticular oligonucleotide agent, and about 1 mg to about 100 mg of thelipid compositions, each per kilogram of patient body weight, isadministered, although higher and lower amounts can be used.

[0260] The agents of the invention are administered to subjects orcontacted with cells in a biologically compatible form suitable forpharmaceutical administration. By “biologically compatible form suitablefor administration” is meant that the oligonucleotide is administered ina form in which any toxic effects are outweighed by the therapeuticeffects of the oligonucleotide. In one embodiment, oligonucleotides canbe administered to subjects. Examples of subjects include mammals, e.g.,humans and other primates; cows, pigs, horses, and farming(agricultural) animals; dogs, cats, and other domesticated pets; mice,rats, and transgenic non-human animals.

[0261] Administration of an active amount of an oligonucleotide of thepresent invention is defined as an amount effective, at dosages and forperiods of time necessary to achieve the desired result. For example, anactive amount of an oligonucleotide may vary according to factors suchas the type of cell, the oligonucleotide used, and for in vivo uses thedisease state, age, sex, and weight of the individual, and the abilityof the oligonucleotide to elicit a desired response in the individual.Establishment of therapeutic levels of oligonucleotides within the cellis dependent upon the rates of uptake and efflux or degradation.Decreasing the degree of degradation prolongs the intracellularhalf-life of the oligonucleotide. Thus, chemically-modifiedoligonucleotides, e.g., with modification of the phosphate backbone, mayrequire different dosing.

[0262] The exact dosage of an oligonucleotide and number of dosesadministered will depend upon the data generated experimentally and inclinical trials. Several factors such as the desired effect, thedelivery vehicle, disease indication, and the route of administration,will affect the dosage. Dosages can be readily determined by one ofordinary skill in the art and formulated into the subject pharmaceuticalcompositions. Preferably, the duration of treatment will extend at leastthrough the course of the disease symptoms.

[0263] Dosage regima may be adjusted to provide the optimum therapeuticresponse. For example, the oligonucleotide may be repeatedlyadministered, e.g., several doses may be administered daily or the dosemay be proportionally reduced as indicated by the exigencies of thetherapeutic situation. One of ordinary skill in the art will readily beable to determine appropriate doses and schedules of administration ofthe subject oligonucleotides, whether the oligonucleotides are to beadministered to cells or to subjects.

[0264] Treatment of Diseases or Disorders

[0265] By inhibiting the expression of a gene, the oligonucleotidecompositions of the present invention can be used to treat any diseaseinvolving the expression of a protein. Examples of diseases that can betreated by oligonucleotide compositions include: cancer, retinopathies,autoimmune diseases, inflammatory diseases (i.e., ICAM-1 relateddisorders, Psoriasis, Ulcerative Colitus, Crohn's disease), viraldiseases (i.e., HIV, Hepatitis C), and cardiovascular diseases.

[0266] In one embodiment, in vitro treatment of cells witholigonucleotides can be used for ex vivo therapy of cells removed from asubject (e.g., for treatment of leukemia or viral infection) or fortreatment of cells which did not originate in the subject, but are to beadministered to the subject (e.g., to eliminate transplantation antigenexpression on cells to be transplanted into a subject). In addition, invitro treatment of cells can be used in non-therapeutic settings, e.g.,to evaluate gene function, to study gene regulation and proteinsynthesis or to evaluate improvements made to oligonucleotides designedto modulate gene expression or protein synthesis. In vivo treatment ofcells can be useful in certain clinical settings where it is desirableto inhibit the expression of a protein. There are numerous medicalconditions for which antisense therapy is reported to be suitable (see,e.g., U.S. Pat. No. 5,830,653) as well as respiratory syncytial virusinfection (WO 95/22,553) influenza virus (WO 94/23,028), andmalignancies (WO 94/08,003). Other examples of clinical uses ofantisense sequences are reviewed, e.g., in Glaser. 1996. GeneticEngineering News 16:1. Exemplary targets for cleavage byoligonucleotides include, e.g., protein kinase Ca, ICAM-1, c-raf kinase,p53, c-myb, and the bcr/abl fusion gene found in chronic myelogenousleukemia.

[0267] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, J. et al. (Cold SpringHarbor Laboratory Press (1989)); Short Protocols in Molecular Biology,3rd Ed., ed. by Ausubel, F. et al. (Wiley, N.Y. (1995)); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed. (1984)); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. (1984)); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); ImmunochemicalMethods In Cell And Molecular Biology (Mayer and Walker, eds., AcademicPress, London (1987)); Handbook Of Experimental Immunology, Volumes I-IV(D. M. Weir and C. C. Blackwell, eds. (1986)); and Miller, J.Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1972)).

[0268] The invention is further illustrated by the following examples,which should not be construed as further limiting.

EXAMPLES Example 1 Oligonucleotide Compositions Comprising ChimericAntisense Sequences

[0269] A gapped antisense oligonucleotide comprising 2′-O-methyl RNAarms and an unmodified DNA gap was synthesized. A complementaryoligonucleotide was also synthesized using unmodified RNA. Adouble-stranded duplex was formed and the composition was found toinhibit expression of the target gene.

Example 2 Length of Double-Stranded ligonucleotides and the Presence orAbsence of Overhangs Has No Effect on Function

[0270] Twenty one and 27-mers were designed to target each of two siteson the p53 molecule (89-90 site, and 93-94 site). The double-strandedmolecules were designed with or without 3′-deoxy TT overhangs. The testoligonucleotides were 21-mers with 2 nucleotide 3′ deoxy TT overhangsand without overhangs (blunt ends); and 27-mers with 2 nucleotide 3′deoxy TT overhangs and without overhangs (blunt ends). Two positivecontrols were included in the experiment (p53) and two negative controlswere also included (FITC)

[0271] A549 cells were transfected with 100 nM of the double-strandedmolecules plus 2 ug/mL Lipofectamine 2000. A549 cells were examined 24hours post-transfection. FITC-labeled molecules were taken up well bycells. Both 21-mers (with or without overhangs) and 27-mers (with orwithout overhangs) were non-toxic to cells. FIG. 1 shows the result ofan experiment comparing the ability of different oligonucleotideconstructs to inhibit p53 and shows that length or the presence orabsence of a 3′ deoxy TT overhang did not affect the activity of theoligonucleotide. The results in FIG. 1 show the amount of p53 mRNAnormalized to the amount of an irrelevant message, GAPDH. The level ofmRNA was determined using RT-PCR analysis. The observed percentinhibition of p53 expression is shown below: 21-MER 27-MER SITE overhangno overhang overhang no overhang 93-94 58% 65% 62% 62% 89-90 81% 75% 67%70%

[0272] Similar results were observed for β-3-integrin; both 21 -mer and27-mer double-stranded molecules were found to inhibit integrin mRNA.Two double-stranded RNA complexes designed to target the same site ofthe β-3-integrin gene were transfected in HMVEC cells. Both complexescontained a two nucleotide (TT) overhang: one complex was a 21-mer (with19 nucleotides complementary to the target gene) and the other was a27-mer (with 25 nucleotides complementary to the target gene). RT-PCRanalysis showed that the two complexes inhibited the target gene to thesame extent. HMVEC cells were transfected using 100 nM oligomercomplexed with 2ug/mL of Lipofecatmine 2000 in media containing serumfor 24 hours. Twenty-four hours after transfection, the cells were lysedand the RNA was isolated for analysis by RT-PCR. No significant toxicitywas observed. The results in FIG. 1B show the amount of β-3-integrinmRNA normalized to the amount of GAPDH, as determined by RT-PCRanalysis.

Example 3 Activation of the Double-Stranded RNA, Interferon-InducibleProtein Kinase, PKR

[0273] PKR is activated by double-stranded RNA molecules. Active PKRleads to the inhibition of protein synthesis, activation oftranscription, and a variety of other cellular effects, including signaltransduction, cell differentiation, cell growth inhibition, apoptosis,and antiviral effects. The effect of p53-targetd double-stranded RNAmolecules on PKR expression was tested. The level of mRNA was determinedusing RT-PCR analysis. As shown in FIG. 2, no correlation was observedbetween the length of the double-stranded oligonucleotide and the levelof PKR induction. Accordingly, long oligonucleotides can be used withoutactivating PKR, a marker for interferon induction.

[0274] As illustrated in FIG. 2B, analysis of relative amounts of PKRmRNA after the 21- and 27-mer transfection in HMVEC cells showedapproximately a 2 fold increase in PKR mRNA of the siRNA controlsequences over no treatment, and approximately a 2 fold increase of PKRmRNA of the 27-mer compared to the 21-mer targeted double-stranded RNAcomplexes.

Example 4 The Effect of 5′ vs. 3′ Modification on the Activity ofDouble-Stranded Oligonucleotides

[0275] Oligonucleotide duplexes were modified at either the 3′ or 5′ endwith FITC groups. The modifications were made on either the antisensestrand or the sense strand. 5′ or 3′ modification of the sense strandhad no effect on the percent inhibition of p53 mRNA. 3′ modification ofthe antisense strand had little affect on activity, while 5′modification of the antisense strand reduced activity significantly. 3′modification of both strands also had little affect on activity, while3′ and 5′ modification of both strands reduced activity. See FIG. 3.

[0276] The effect of the size of the group used to modify the 5′ end wastested. The results of this experiment are shown in FIG. 4. Theinclusion of a 5′ phosphate group had little affect on activity, whereasthe modification of the antisense strand or both strands had a greatereffect. The inclusion of a propyl group had more of an effect, with a 5′propyl group on the antisense strand showing a large reduction inactivity; there was also an effect when this group was added to bothstrands. Similarly, the inclusion of a FITC group at the 5′ end of theantisense molecule (or to both molecules) also significantly reduced theactivity of the RNA duplex.

Example 5 Comparison of the Efficacy of 2′-O-Me Modified and UnmodifiedDouble-Stranded RNA Oligonucleotides

[0277] A549 cells were transfected with modified or unmodified RNAduplexes complexed at 100 nM with 2 ug/mL Lipofectamine 2000(Invitrogen) and were transfected for 24 hours. The A549 cells wereplated at 20,000/well in 48 well plates. After 24 hours, FITC-labeleddouble-stranded oligonucleotides were visible in A549 cells; theinclusion of a 2′-O-Me group did not affect uptake. The Table belowshows the results of this experiment. 2′-O-Me Oligonucleotide DuplexesAnti- sense/Sense Anti- Anti- Anti-sense/ 2′-O-Me/2′-O- sense/Sensesense/Sense Sense Me 2′-O-Me/RNA RNA/2′-O-Me RNA/RNA targeted18639/18640 18639/16194 16193/18640 18876 non- 19039/19040 19039/1904419043/19040 18850 & targeted 16197/16198 FITC-2′-O-Me/ FITC-2′-O-Me/FITC-2′-O-Me/ 2′-O-Me/ FITC 2′-O-Me FITC-RNA RNA FITC-RNA non- 1920919037/19042 19037/19044 19039/19042 targeted

[0278] The affect of 2′-O-Me modifications to one or both strands of adouble-stranded RNA molecule is shown in FIG. 5.

Example 6 Toxicity of p53-Targeted siRNAs in A549 Cells

[0279] 27-mer siRNAs targeting p53 were not toxic to cells when comparedto standard 21-mer siRNAs having 3′ deoxy TT overhangs. In thisexperiment, both siRNA constructs inhibited p53 to a similar extent (83%inhibition for 27-mer vs. 90% inhibition for 21-mer). siRNAs weredesigned to target p53 and were constructed as blunt-end 27-mers or as21-mers with 3′ deoxy TT overhangs. A549 cells were plated at 20,000cells per well in 48-well plates on the day prior to transfection. Onthe day of transfection, cells were approximately 60-70% confluent.Cells were transfected with 100 nM siRNAs complexed with 2 ug/mLLipofectamine 2000 for 24 hours. Following transfection, cells werestained with Dead Red stain to visualize the extent of cell death. ThesiRNA sequences used were as follows: 21-mer with overhangs targeted(5′-3′): ACCUCAAAGCUGUUCCGUCTT (SEQ ID NO: ##) GACGGAACAGCUUUGAGGUTT(SEQ ID NO: ##) Blunt-end 27-mer targeted (5′-3′):ACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: ##) GGGACGGAACAGCTTTGAGGTGTGCGT(SEQ ID NO: ##)

Example 7 Toxicity of Blunt-End 27-mer siRNAs Targeting p53 in A549Cells

[0280] The toxicity of targeted blunt-end 27-mer siRNAs targeting p53was observed to be not significantly different than a control nucleicacid or no treatment. siRNAs were designed to target p53 and wereconstructed as blunt-end 27-mers. The corresponding control consisted ofchemistry-matched, scrambled sequences with a similar base-paircomposition. A549 cells were plated at 20,000 cells per well in 48-wellplates on the day prior to transfection. On the day of transfection,cells were approximately 60-70% confluent. Cells were transfected with100 nM siRNAs complexed with 2 ug/mL Lipofectamine 2000 for 24 hours.Following transfection, the cells were stained with Dead Red stain tovisualize the extent of cell death. The siRNA sequences used were asfollows: Blunt-end 27-mer targeted (5′-3′ on top):ACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: ##) GGGACGGAACAGCTTTGAGGTGTGCGT(SEQ ID NO: ##) Corresponding control (5′-3′ on top):CCCTGCCTTGTCGAAACTCCACACGCA (SEQ ID NO: ##) TGCGTGTGGAGTTTCGACAAGGCAGGG(SEQ ID NO: ##)

Example 8 Toxicity of Blunt End 32-mer siRNAs Targeting p53 in A549Cells

[0281] Similarly, blunt-end 32-mer siRNAs targeting p53 were notobserved to be toxic to cells in comparison with a control nucleic acidand no treatment, as determined by Dead Red staining. siRNAs weredesigned to target p53 and were constructed as blunt-end 32-mers. Thecorresponding control consisted of chemistry-matched, scrambledsequences with a similar base-pair composition. A549 cells were platedat 20,000 cells per well in 48-well plates on the day prior totransfection. On the day of transfection, cells were approximately60-70% confluent. Cells were transfected with 100 nM siRNAs complexedwith 2 ug/mL Lipofectamine 2000 for 24 hours. Following transfection,cells were stained with Dead Red stain to visualize the extent of celldeath. The siRNA sequences used were as follows: Targeted blunt-end32-mer (5′-3′ on top:) CCCTCACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: ##)GGGACGGAACAGCTTTGAGGTGTGCGTGAGGG (SEQ ID NO: ##) Corresponding control(5′-3′ on top): CCCTGCCTTGTCGAAACTCCACACGCACTCCC (SEQ ID NO: ##)GGGAGTGCGTGTGGAGTTTCGACAAGGCAGGG (SEQ ID NO: ##)

Example 9 Inhibition of p53 by 32- and 37-mer Blunt-End siRNAs

[0282]FIG. 6 depicts the results of inhibition of p53 by 32- and 37-merblunt-end siRNAs in comparison with various control experiments. siRNAswere designed to target each of two sites (93-93 site) and (89-90 site)along the coding region of p53. siRNAs were constructed as blunt-end32-mers or blunt-end 37-mers. Positive control siRNAs were 21-mers with3′ deoxy TT overhangs. Corresponding controls consisted ofchemistry-matched, scrambled sequences with a similar base-paircomposition. A549 cells were plated at 20,000 cells per well in 48-wellplates on the day prior to transfection. On the day of transfection,cells were approximately 60-70% confluent. Cells were transfected with100 nM siRNAs complexed with 2 ug/mL Lipofectamine 2000 for 24 hours.Following transfection, cells were lysed and poly(A) mRNA was harvestedfor RT-PCR. Inhibition of p53 expression was determined by quantitativereal-time RT-PCR (TaqMan) analysis. Expression of p53 was standardizedby quantifying GAPDH for each sample. The data in FIG. 6 represent threeseparate transfections analyzed in duplicate and normalized to theinternal control (GAPDH). The siRNA sequences used were as follows(depicted with the 5′-3′ strand on top): Targeted 32-mer (89-90 site):CCCTCACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: ##)GGGACGGAACAGCTTTGAGGTGTGCGTGAGGG (SEQ ID NO: ##) 32-mer control (89-90site): CCCTGCCTTGTCGAAACTCCACACGCACTCCC (SEQ ID NO: ##)GGGAGTGCGTGTGGAGTTTCGACAAGGCAGGG (SEQ ID NO: ##) 32-mer targeted (93-94site): CCCUUCUGUCUUGAACAUGAGTTTTTTATGGC (SEQ ID NO: ##)GCCATAAAAAACTCATGTTCAAGACAGAAGGG (SEQ ID NO: ##) 32-mer control (93-94site): CGGTATTTTTTGAGTACAAGTTCTGTCTTCCC (SEQ ID NO: ##)GGGAAGACAGAACTTGTACTCAAAAAATACCG (SEQ ID NO: ##) 37-mer targeted (93-94site): CCCTTCTGTCTTGAACATGAGTTTTTTATGGCGGGAG (SEQ ID NO: ##)CTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGG (SEQ ID NO: ##) 37-mer control(93-94 site): GAGGGCGGTATTTTTTGAGTACAAGTTCTGTCTTCCC (SEQ ID NO: ##)GGGAAGACAGAACTTGTACTCAAAAAATACCGCCCTC (SEQ ID NO: ##) 21-mer targeted(89-90 site): ACCUCAAAGCUGUUCCGUCTT (SEQ ID NO: ##)GACGGAACAGCUUUGAGGUTT (SEQ ID NO: ##) 21-mer targeted (93-94 site):CCCUUCUGUCUUGAACAUGTT (SEQ ID NO: ##) CAUGUUCAAGACAGAAGGGTT (SEQ ID NO:##)

Example 10 Enhanced Cellular Stability of Double-Stranded 2′-O-MethylRNA

[0283] In this example, the single-stranded control oligomer wastransfected at 800 nM. Accumulation was observed in the nucleus at 6hours post transfection, however by 25 hours the fluorescence of thesingle-stranded oligomer had largely dissipated, indicating the oligomerwas no longer intact (Fisher, T., T. Terhorst, et al. (1993).“Intracellular disposition and metabolism of fluorescently-labeledunmodifieed and modified oligonucleotides microinjected into mammaliancells.” NAR 21: 3857-3865). The relative fluorescence offluorescently-labeled oligomers transfected into A549 cells was observedto fit the following pattern: single-stranded (800 nM) double-stranded(100 nm)  6 h ++++ +++++ 25 h + +++++

[0284] The double-stranded oligomer duplex, wherein the second strandwas 2′-O-methyl modified RNA, was transfected at 100 nM, and was alsoclearly visible at 6 hours post transfection. However, in contrast tothe single-stranded oligomer, the double-stranded was still largelyintact in the nucleus at 24 hours, even though the concentrationtransfected was 8-fold less, thereby demonstrating that the 2′-O-methylsecond strand stabilized the oligomer in the cell.

[0285] The oligomers were all 2′-O-CH₃ with a phosphodiester backbonecontaining 6-carboxyfluorescein (6-FAM) tethered to the 5′ hydroxyl. Thesingle-stranded control oligomer was transfected at 800 nM complexedwith 4 ug/mL of Lipofectamine 2000, and the double-stranded complex wastransfected at 100 nM complexed with 1 ug/mL of Lipofectamine 2000.

[0286] Fluorescent signal was seen accumulating in the nucleus at 6hours post transfection, however by 24 hours the single-strandedoligomer has significantly dissipated, indicating the oligomer is nolonger intact. The double-stranded duplexes (wherein the second strandis 2′-O-methyl modified RNA with a 5′ 6-FAM) was transfected at 100 nM,and was also clearly visible at 6 hours post transfection. In contrastto the single-stranded oligomer, the double-stranded was still largelyintact in the nucleus at 24 hours, even though the concentrationtransfected was 8-fold less. This experiment demonstrates that the2′-O-methyl second strand stabilizes the duplex in the cell.

Example 11 Enhanced Stability in Cells and Accumulation in Cytoplasm ofRNA Hybridized to 2′-O-Methyl RNA

[0287] The fluorescence signal, corresponding to uptake of FITC-labeledRNA and 2′-O-methyl modified RNA duplexes, was measured at 6 and 24hours. RNA complexes were transfected in A549 cells with 100 nM oligomercomplexed with 2 ug/mL Lipofecatmine 2000 as described below. Cells werecontinuously transfected for 24 hours and fluorescent uptake wasassessed at 6 and 24 hours. Oligomers were 2′-O-methyl modified RNA with5′ 6-FAM (FITC-2′-OMe), 19-mer RNA with two deoxynucleotides on the 3′end with 5′ 6-FAM (FITC-RNA) or 19-mer RNA with two deoxynucleotides onthe 3′ end (RNA) complexed. At 6 hours, the FITC-2′-O-methyl duplexesshow localization in the nucleus and the FITC-2′-O-methyl/RNA and2′-O-methyl/FITC-RNA complexes show a more diffuse pattern of uptake(these RNA/2′-O-methyl complexes are a substrate for the RISC complexand are therefore retained in the cytoplasm where the RISC complex hasbeen reported to be active). At 24 hours, the FITC-2′-O-methyl/RNA and2′-O-methyl/FITC-RNA complexes were still visible in the cell, whereastypically not even the single-stranded FITC-2′-O- was visible, even whentransfected at significantly higher concentrations, demonstrating thatthe 2′-O-methyl RNA protects the RNA strand from degradation in thecell.

[0288] RNA oligomers having a phosphodiester backbone with 2′-O-methylnucleotides were synthesized using standard phosphoramidite chemistry.Oligomers were purified by denaturing polyacrylamide gel electrophoresis(PAGE). Purity of oligomers was confirmed by (PAGE) and massspectrometry. All oligomers were greater than 90% full length, and massdata obtained was consistent with expected values. Target-specific siRNAduplexes consisted of 21-nt sense and 21-nt antisense strands withsymmetric 2-nt 3′ deoxy TT overhangs. 21-nt RNAs were chemicallysynthesized using phosphoramidite chemistry. For duplex preparation,sense- and antisense oligomers (each at 50 μM) were combined in equalvolumes in annealing buffer (30 mM HEPES pH 7.0, 100 mM potassiumacetate, and 2 mM magnesium acetate), heat-denatured at 90° C. for 1 minand annealed at 37° C. for one hour. Duplexes were stored at 80° C.until used.

[0289] A549 cells (ATCC #CCL-185) were cultured at 37° C. in Dulbecco'sModified Eagle Medium (DMEM, Life Technologies #11960-044) supplementedwith 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin,and 10% fetal bovine serum (FBS). HeLa cells (ATCC #CCL-2) were culturedat 37° C. in Minimal Essential Medium (MEM, Life Technologies#10370-021) supplemented with 2 mM L-glutamine, 1.5 g/L sodiumbicarbonate, 1.0 mM sodium pyruvate, 100 units/mL penicillin, 100 μg/mLstreptomycin, and 10% FBS. Cells were passaged regularly to maintainexponential growth. On the day prior to transfection, cells weretrypsinized, counted, and seeded in 48-well plates at a density of20×103 cells per well in 250 μL fresh media. On the day of transfectioncells were typically 60-65% confluent. Transfection of siRNA duplexesand oligomers was carried out using Lipofectamine 2000 (LifeTechnologies). Briefly, a 10× stock of Lipofectamine 2000 was preparedin Opti-Mem (Life Technologies) and incubated at room temperature for 15minutes. An equal volume of a 10× stock of siRNA duplex or oligomers inOpti-Mem was added and complexation carried out for 15 minutes at roomtemperature. Complexes were then diluted 5-fold in full growth media.Culture media was removed from each well prior to the addition of 250 μLcomplexes per well. Cells were incubated at 37° C./5% CO₂ for 6 or 24hours prior to assessing the uptake.

[0290] Equivalents

[0291] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims. The entirecontents of all patents, published patent applications and otherreferences cited herein are hereby expressly incorporated herein intheir entireties by reference.

1. A double-stranded oligonucleotide composition having the structure:

wherein (1) n is a nucleomonomer in complementary oligonucleotidestrands of equal length and where the sequence of Ns corresponds to atarget gene sequence and (2) X and Y are each independently selectedfrom a group consisting of nothing; from about 1 to about 20 nucleotidesof 5′ overhang; from about 1 to about 20 nucleotides of 3′ overhang; anda loop structure consisting from about 4 to about 20 nucleomonomers,where the nucleomonomers are selected from the group consisting of G andA.
 2. A double-stranded oligonucleotide composition having thestructure:

wherein (1) oligoA is an oligonucleotide of a number of nucleomonomers;(2) oligoB is an oligonucleotide that has the same number ofnucleomonomers as oligoA and that is complementary to oligoA; (3) eitheroligoA or oligoB corresponds to a target gene sequence; (4) X isselected from a group consisting of (a) nothing; (b) an oligonucleotideof about 1 to about 20 nucleotides covalently bonded to the 5′ end ofoligoA and constituting a 5′ overhang; (c) an oligonucleotide of about 1to about 20 nucleotides covalently bonded to the 3′ end of oligoB andconstituting a 3′ overhang; (d) and an oligonucleotide of about 4 toabout 20 nucleomonomers covalently bonded to the 3′ end of oligoB andthe 5′ end of oligoA and constituting a loop structure, where thenucleomonomers are selected from the group consisting of G and A and (5)Y is selected from a group consisting of (a) nothing; (b) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 5′ end of oligoB and constituting a 5′ overhang; (c) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 3′ end of oligoA and constituting a 3′ overhang; (d) and anoligonucleotide of about 4 to about 20 nucleomonomers covalently bondedto the 3′ end of oligoA and the 5′ end of oligoB and constituting a loopstructure, where the nucleomonomers are selected from the groupconsisting of G and A.
 3. The composition of claim 1, wherein the numberof nucleomonomers in each strand of the duplex is between about 12 andabout
 40. 4. The composition of claim 2, wherein the number ofnucleomonomers in each strand oligoA and oligoB is between about 12 andabout
 40. 5. The composition of claim 1, wherein the number ofnucleomonomers in each strand of the duplex is about
 27. 6. Thecomposition of claim 2, wherein the number of nucleomonomers in eachstrand of oligoA and oligoB is about
 27. 7. The composition of claim 1,wherein X is a sequence of about 4 to about 20 nucleomonomers which forma loop, wherein the nucleomonomers are selected from the groupconsisting of G and A.
 8. The composition of claim 2, wherein X or Y isa sequence of about 4 to about 20 nucleomonomers that forms a loop,wherein the nucleomonomers are selected from the group consisting of Gand A.
 9. The composition of claim 8, wherein two of the adjacent Ns areunlinked.
 10. The composition of claim 8, wherein the nucleotidesequence of the loop is GAAA.
 11. A double-stranded oligonucleotidecomposition having the structure: 5′-(Z)₂₋₈-(N)₁₅₋₄₀-(M)₂₋₈-3′3′-(Z)₂₋₈-(N)₁₅₋₄₀-(M)₂₋₈-5′

wherein (1) each of N, Z, and M is independently a nucleomonomer; (2)both of the sequences of Ns are complementary oligonucleotide strands ofequal length having between about 15 and about 40 nucleomonomers; (3) atleast one of the sequences of Ns, optionally with some or all of theflanking Ms or Zs, corresponds to a target gene sequence; (4) both ofthe sequences of Zs are complementary oligonucleotide strands of betweenabout 2 and about 8 nucleomonomers in length; and (5) both of thesequences of Ms are complementary oligonucleotide strands of betweenabout 2 and about 8 nucleomonomers in length.
 12. The composition ofclaim 11, wherein each Z and M nucleomonomer is selected from the groupconsisting of C and G.
 13. The composition of claim 12 wherein, thesequence of Zs or Ms is CC, GG, CG, GC, CCC, GGG, CGG, GCC, GCG, CGC,CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC, orCCGG.
 14. A double-stranded oligonucleotide composition having thestructure:

wherein (1) N is a nucleomonomer in complementary oligonucleotidestrands of equal length and where the sequence of Ns corresponds to atarget gene sequence and (2) X is selected from the group consisting ofnothing; 1-20 nucleotides of 5′ overhang; 1-20 nucleotides of 3′overhang; a loop structure consisting of from about 4 to about 20nucleomonomers, where the nucleomonomers are selected from the groupconsisting of G and A, and (3) where M is a nucleomonomer incomplementary oligonucleotide strands of between about 2 and about 8nucleomonomers in length which optionally correspond to the targetsequence.
 15. A double-stranded oligonucleotide composition having thestructure:

wherein (1) oligoA is 5′-(N)₁₅₋₄₀-(M)₂₋₈-3′ and oligoB is5′-(N)₁₅₋₄₀-(M)₂₋₈-3′, wherein each of N and M is independently anucleomonomer; (2) both of the sequences of Ns are complementaryoligonucleotide strands of equal length having between about 15 and 40nucleomonomers; (3) at least one of the sequences of Ns, optionally withsome or all of the flanking Ms, corresponds to a target gene sequence;(4) X is selected from a group consisting of (a) nothing; (b) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 5′ end of oligoA and constituting a 5′ overhang; (c) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 3′ end of oligoB and constituting a 3′ overhang; (d) and anoligonucleotide of about 4 to about 20 nucleomonomers covalently bondedto the 3′ end of oligoB and the 5′ end of oligoA and constituting a loopstructure, where the nucleomonomers are selected from the groupconsisting of G and A; and (5) both of the sequences of Ms arecomplementary oligonucleotide strands of between about 2 and about 8nucleomonomers in length.
 16. The composition of claim 15, wherein Mnucleomonomer is selected from the group consisting of contain C and G.17. The composition of claim 16, wherein the sequence of M is CC, GG,CG, GC, CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC,CGCG, GGGC, CCCG, CGGG, GCCC, GGCC, or CCGG.
 18. A double-strandedoligonucleotide composition having the structure:

wherein (1) N is a nucleomonomer in complementary oligonucleotidestrands of equal length and which correspond to a target gene sequenceand (2) Y is selected from the group consisting of nothing; 1-20nucleotides of 5′ overhang; 1-20 nucleotides of 3′ overhang; a loopconsisting of a sequence of from about 4 to about 20 nucleomonomers,where the nucleomonomers are all either Gs or A's and (3) where Z is aare nucleomonomer in complementary oligonucleotide strands of betweenabout 2 and about 8 nucleomonomers in length and which comprise asequence which can optionally correspond to the target sequence.
 19. Adouble-stranded oligonucleotide composition having the structure:

wherein (1) oligoA is 5′-(Z)₂₋₈-(N)₁₂₋₄₀-3′ and oligoB is5′-(Z)₂₋₈-(N)₁₂₋₄₀-3′, wherein each of N and Z is independently anucleomonomer; (2) both of the sequences of Ns are complementaryoligonucleotide strands of equal length having between about 12 and 40nucleomonomers; (3) at least one of the sequences of Ns, optionally withsome or all of the flanking Zs, corresponds to a target gene sequence;(4) Y is selected from a group consisting of (a) nothing; (b) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 5′ end of oligoB and constituting a 5′ overhang; (c) anoligonucleotide of about 1 to about 20 nucleotides covalently bonded tothe 3′ end of oligoA and constituting a 3′ overhang; (d) and anoligonucleotide of about 4 to about 20 nucleomonomers covalently bondedto the 3′ end of oligoA and the 5′ end of oligoB and constituting a loopstructure, where the nucleomonomers are selected from the groupconsisting of G and A; and (5) both of the sequences of Zs arecomplementary oligonucleotide strands of between about 2 and about 8nucleomonomers in length.
 20. The composition of claim 19, wherein the Znucleomonomers are selected from the group consisting of C and G. 21.The composition of claim 20, wherein the sequence of Z is CC, GG, CG,GC, CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG,GGGC, CCCG, CGGG, GCCC, GGCC, or CCGG.
 22. A method of regulating geneexpression in a cell, comprising contacting a cell with thedouble-stranded duplex oligonucleotide composition of claim 1, tothereby regulate gene expression in a cell.
 23. A method of increasingthe nuclease resistance of an antisense sequence, comprising forming adouble-stranded oligonucleotide composition of claim 1, such that adouble-stranded duplex is formed, wherein the nuclease resistance of theantisense sequence is increased compared to a control composition.